MAIN  LI-BRAHY-AGRFCULTUr-E  D 


BIOLOCJY 

LIBRARY 

G 


MANUAL   OF   BACTERIOLOGY 


MANUAL 


OF 


BACTERIOLOGY 


BY 


ROBERT    MUIR,    M.A.,  M.D.,  F.R.C.P.ED. 

PROFESSOR   OF   PATHOLOGY,    UNIVERSITY   OF   GLASGOW, 


AND 


JAMES    RITCHIE,    M.A.,  M.D.,  B.Sc. 

READER   IN   PATHOLOGY,    UNIVERSITY   OF   OXFORD. 


AMERICAN   EDITION    (WITH    ADDITIONS), 

REVISED   AND   EDITED   FROM   THE   THIRD    ENGLISH    EDITION 

BY 

NORMAN    MAC   LEOD    HARRIS,   M.B.  (TOR.) 

ASSOCIATE   IN   BACTERIOLOGY,  THE  JOHNS   HOPKINS   UNIVERSITY,   BALTIMORE. 


WITH  ONE  HUNDRED  &  SEVENTY  ILLUSTRATIONS. 


gnrfe: 
THE    MACMILLAN   COMPANY, 

LONDON:   MACMILLAN  &  CO.,   LTD. 
1903 

jill  rights  reserved. 


HD3 


BIOLOGt 

LIBRARY 

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Main 


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COPYRIGHT,  1903, 
BY  THE  MACMILLAN   COMPANY. 


Set  up  and  electrotyped  February,  1903. 


Norwood  Press 

J.  S.  Gushing  &  Co.  —  Berwick  &  Smith 
Norwood,  Mass.,  U.S. A, 


PREFACE    TO    THE    AMERICAN    EDITION. 

IN  presenting  this  the  American  edition  of  the  well-known 
and  appreciated  work  of  Doctors  Muir  and  Ritchie,  the  en- 
deavour has  been  made  to  add  to  the  value  of  the  book  by 
giving  adequate  expression  to  the  best  in  American  laboratory 
methods  and  research,  and,  at  the  same  time,  to  augment  the 
general  scope  of  the  work  without  eliminating  the  personal 
impress  of  the  authors.  Therefore  occasional  alterations  and 
additions  of  greater  or  lesser  magnitude  have  been  made 
throughout  the  book  in  general,  but  more  especially  in  Chap- 
ters II,  III,  IV,  XV,  and  XVII;  whilst  the  chapter  on  Fungi, 
deleted  from  the  last  English  edition,  has  been  retained  and 
enlarged.  The  section  of  the  Manual  dealing  with  the  bibli- 
ography has  also  been  extended  to  cover  as  far  as  possible  the 
advances  in  work  made  in  this  country  and  abroad. 

Some  photographic  reproductions  and  a  few  engravings  of 
apparatus  have  been  added  to  those  of  the  English  edition,  for 
which  hearty  thanks  is  due  Doctors  A.  C.  Abbott,  T.  C.  Gil- 
christ,  and  Charles  Potter,  and  Messrs.  Charles  Lentz  &  Sons, 
and  W.  B.  Saunders  &  Co.  for  their  kindness  in  furnishing  the 
same. 

The  Editor  takes  this  opportunity  of  expressing  his  in- 
debtedness to  Professor  William  H.  Welch  for  many  helpful 
suggestions  in  the  preparation  of  the  edition. 


N.  MAC  L.  H. 


THE  JOHNS  HOPKINS  UNIVERSITY, 

BALTIMORE,  MARYLAND, 

November,  1902. 


112895 


PREFACE   TO    THE    THIRD    EDITION. 

IN  this  edition  the  whole  subject  has  been  carefully  revised, 
and,  as  formerly,  we  have  aimed  at  making  the  bearings  of 
bacterial  action  on  general  pathological  processes  an  outstand- 
ing feature. 

The  advances  in  bacteriology  during  the  last  three  years 
have  been  neither  few  nor  of  small  importance,  and  to  incor- 
porate these  and  at  the  same  time  maintain  the  work  as  a 
convenient  hand-book  for  the  student,  has  been  no  easy  task. 
In  endeavouring  to  accomplish  it,  we  have  condensed  various 
portions  and  omitted  others  which  appear  now  to  be  of  sub- 
sidiary importance.  Thus,  although  much  new  matter  has  been 
introduced,  the  former  length  of  the  volume  has  been  bi  t 
slightly  increased.  'Additions  have  been  made  to  most  of  tho 
chapters  and  several  new  subjects  are  treated  of,  amongst 
which  may  be  mentioned  the  bacteriology  of  the  air,  soil,  and 
water,  to  which  a  new  chapter  has  been  devoted.  The  chapter 
on  Immunity  has  been  modified  and  extended  so  as  to  include 
the  recent  important  researches  on  the  subject.  A  number  of 
new  illustrations  will  be  found  to  have  been  added,  and  we 
trust  that  these  will  tend  towards  the  elucidation  of  the  text. 

One  result  of  later  research  in  bacteriology  has  been  to 
bring  into  prominence  the  fact  that,  in  nearly  every  instanee, 
each  so-called  pathogenic  organism  is  a  member  of  a  group  of 
bacteria  possessing  closely  allied  characters.  Hard  and  fast 
lines  as  to  distinguishing  features  can  now  be  less  definitely 
drawn,  and  accordingly  an  intelligent  conception  on  the  part 
of  the  student  is  more  than  ever  necessary.  We  have  there- 
fore in  many  instances  merely  stated  the  known  facts,  when 
we  have  considered  that  these  do  not  justify  an  advance  being 
made  to  a  definite  conclusion. 

JANUARY,  1903. 


vi 


PREFACE  TO  SECOND  EDITION. 

IN  preparing  this  edition  we  have  made  no  change  in  the 
original  plan  of  the  book.  The  text,  however,  has  been  care- 
fully revised,  and  the  results  of  the  more  recent  researches 
have  been  incorporated.  Some  parts  have  been  condensed, 
but,  in  consequence  of  the  introduction  of  new  subject-matter 
and  of  additional  illustrations,  the  size  of  the  book  as  a  whole 
has  been  considerably  increased.  We  trust  that  these  altera- 
tions will  be  found  to  be  in  the  direction  of  improvement. 

MAY,  1899. 


vii 


PREFACE   TO   THE   FIRST   EDITION. 

THE  science  of  Bacteriology  has,  within  recent  years,  be- 
come so  extensive,  that  in  treating  the  subject  in  a  book  of  this 
size  we  are  necessarily  restricted  to  some  special  departments, 
unless  the  description  is  to  be  of  a  superficial  character.  Ac- 
cordingly, as  this  work  is  intended  primarily  for  students  and 
practitioners  of  medicine,  only  those  bacteria  which  are  asso- 
ciated with  disease  in  the  human  subject  have  been  considered. 
We  have  made  it  a  chief  endeavour  to  render  the  work  of 
practical  utility  for  beginners,  and,  in  the  account  of  the  more 
important  methods,  have  given  elementary  details  which  our 
experience  in  the  practical  teaching  of  the  subject  has  shown 
to  be  necessary. 

In  the  systematic  description  of  the  various  bacteria,  an 
attempt  has  been  made  to  bring  into  prominence  the  evidence 
of  their  having  an  etiological  relationship  to  the  corresponding 
diseases,  to  point  out  the  general  laws  governing  their  action 
as  producers  of  disease,  and  to  consider  the  effects  in  particular 
instances  of  various  modifying  circumstances.  Much  research 
on  certain  subjects  is  so  recent  that  conclusions  on  many  points 
must  necessarily  be  of  a  tentative  character.  We  have,  there- 
fore, in  our  statement  of  results  aimed  at  drawing  a  distinction 
between  what  is  proved  and  what  is  only  probable. 

In  an  Appendix  we  have  treated  of  four  diseases ;  in  two  of 
these  the  causal  organism  is  not  a  bacterium,  whilst  in  the 
other  two  its  nature  is  not  yet  determined.  These  diseases 
have  been  included  on  account  of  their  own  importance  and 
that  of  the  pathological  processes  which  they  illustrate. 

Our  best  thanks  are  due  to  Professor  Greenfield  for  his  kind 
advice  in  connection  with  certain  parts  of  the  work.  We  have 
also  great  pleasure  in  acknowledging  our  indebtedness  to  Dr. 
Patrick  Manson,  who  kindly  lent  us  the  negatives  or  prepara- 
tions from  which  Figs.  143-148  have  been  executed. 


PREFACE   TO   THE   FIRST  EDITION.  ix 

As  we  are  convinced  that  to  any  one  engaged  in  practical 
study,  photographs  and  photomicrographs  supply  the  most 
useful  and  exact  information,  we  have  used  these  almost  exclu- 
sively in  illustration  of  the  systematic  description.  These  have 
been  executed  in  the  Pathological  Laboratory  of  the  University 
of  Edinburgh  by  Mr.  Richard  Muir.  The  line  drawings  were 
prepared  for  us  by  Mr.  Alfred  Robinson,  of  the  University 
Museum,  Oxford. 

To  the  volume  is  appended  a  short  Bibliography,  which, 
while  having  no  pretension  to  completeness,  will,  we  hope,  be 
of  use  in  putting  those  who  desire  further  information  on  the 
track  of  the  principal  papers  which  have  been  published  on 
each  of  the  subjects  considered. 

JUNE,  1897. 


CONTENTS. 

CHAPTER   I. 
GENERAL  MORPHOLOGY  AND  BIOLOGY. 

PAGE 

Introductory  —  Terminology  —  Structure  of  the  bacterial  cell  —  Reproduction 
of  bacteria  —  Spore  formation  —  Motility  —  Minuter  structure  of  bacterial 
protoplasm  —  Chemical  composition  of  bacteria  —  Classification  —  Food 
supply — Relation  of  bacteria  to  moisture,  gaseous  environment,  tempera- 
ture, and  light  —  Conditions  affecting  bacterial  motility  —  Effects  of  bac- 
teria in  nature  —  Methods  of  bacterial  action  —  Variability  among  bacteria  I 

CHAPTER   II. 
METHODS  OF  CULTIVATION  OF  BACTERIA. 

Introductory  —  Methods  of  sterilisation  —  The  preparation  of  culture  media  — 
The  use  of  the  culture  media  —  The  methods  of  the  separation  of  aerobic 
organisms  —  The  principles  of  the  culture  of  anaerobic  organisms  —  Mis- 
cellaneous methods  —  General  laboratory  rules 27 

CHAPTER    III.. 

MICROSCOPIC  METHODS — GENERAL  BACTERIOLOGICAL  DIAGNOSIS 
—  INOCULATION  OF  ANIMALS. 

The  microscope  —  Examination  of  hanging-drop  cultures — Film  preparations 

—  Examination  of  bacteria  in  tissues  —  The  cutting  of  sections — Staining 
principles  —  Mordants   and   decolorisers  —  Formulae   of  stains  —  Gram's 
method  and  its  modifications  —  Stain  for  tubercle   and  leprosy  bacilli  — 
Staining  of  spores  and  flagella  —  Observation  of  agglutination  and  sedi- 
mentation —  Routine  bacteriological  examination  —  Methods  of  inoculation 

—  Autopsies  on  animals 87 

CHAPTER    IV. 
BACTERIA  IN  AIR,  SOIL,  AND  WATER.    ANTISEPTICS. 

AIR:  Methods  of  examination  —  Results.  SOIL:  Methods  of  examination  — 
Varieties  of  bacteria  in  soil.  WATER  :  Methods  of  examination  —  Bacteria 
in  water  —  Bacterial  treatment  of  sewage.  ANTISEPTICS  :  Methods  of 
investigation  —  The  action  of  antiseptics  —  Certain  particular  antiseptics  .  123 


xii  CONTENTS. 

CHAPTER   V. 
FUNGI:   NON-PATHOGENIC  AND  PATHOGENIC. 

PAGE 

Mucorinae  —  Ascomycetse —  Perisporiaceae  —  Yeasts  and  torulse  —  Blastomycetic 

dermatitis — Morphology  —  Cultural  characters 148 

CHAPTER   VI. 

RELATIONS  OF  BACTERIA  TO  DISEASE  —  THE  PRODUCTION  OF 
TOXINS  BY  BACTERIA. 

Introductory  —  Conditions  modifying  pathogenicity  —  Modes  of  bacterial  action 

—  Tissue  changes  produced  by  bacteria  —  Local  lesions  —  General  lesions 

—  Disturbance  of  metabolism  by  bacterial  action  —  The   production  of 
toxins  by  bacteria,  and  the  nature  of  these  —  Allied  vegetable  and  animal 
poisons — The  theory  of  toxic  action 157 

CHAPTER    VII. 
INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS. 

The  relations  of  inflammation  and  suppuration  —  The  bacteria  of  inflammation 
and  suppuration  —  Results  of  experimental  inoculation  —  Lesions  in  the 
human  subject  —  Mode  of  entrance  and  spread  of  pyogenic  bacteria  — 
Ulcerative  endocarditis  —  Acute  suppurative  periostitis  —  Erysipelas  — 
Meningitis  —  Conjunctivitis  —  Acute  rheumatism  —  Methods  of  examina- 
tion in  inflammatory  and  suppurative  conditions 180 

CHAPTER   VIII. 

INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS,  CONTINUED: 
THE  ACUTE  PNEUMONIAS. 

Introductory  —  Historical  —  Bacteria  in  pneumonia  —  Fraenkel's  Pneumococ- 
cus  —  Friedlander's  pneumobacillus  —  Distribution  of  pneumobacteria  — 
Experimental  inoculation  —  Etiological  relations  to  pneumonia  —  The 
toxins  of  the  pneumococcus  —  Immunisation  against  pneumococcus  — 
Methods  of  examination 205 

CHAPTER   IX. 
GONORRHOEA,  SOFT  SORE,  SYPHILIS. 

The  gonococcus  —  Microscopical  characters  —  Cultivation  —  Relations  to 
disease  —  Its  toxin  —  Distribution  —  Gonococcus  in  joint  affections  — 
Methods  of  diagnosis  —  Soft  sore  —  Syphilis 223 

CHAPTER   X. 
TUBERCULOSIS. 

Historical — Tuberculosis  in  animals  —  The  tubercle  bacillus  —  Staining  reac- 
tions—  Cultivation  of  tubercle  bacillus  —  Powers  of  resistance  —  Action 
on  the  tissues  —  Histology  of  tuberculous  nodules — Distribution  of  bacilli 


CONTENTS.  xiii 


—  Bacilli  in  tuberculous  discharges  —  Experimental  inoculation  —  Varieties 
of  tuberculosis  —  Other  acid-fast  bacilli  —  Action  of  dead  tubercle  bacilli 

—  Sources  of  human  tuberculosis  —  Koch's  tuberculin  —  Toxins  of  the 
tubercle  bacillus  —  Koch's  new  tuberculin  —  Immunisation  against  tubercle 

—  Methods  of  examination          .         .         .    . 236 

CHAPTER  XL 
LEPROSY. 

Pathological  changes  —  Bacillus  of  leprosy  —  Distribution  in  lesions  —  Rela- 
tions to  disease  —  Methods  of  diagnosis 267 

CHAPTER  XII. 
GLANDERS  AND  RHINOSCLEROMA. 

The  natural  disease  —  The  bacillus  of  glanders  —  Cultivation  of  glanders 
bacillus  —  Powers  of  resistance  —  Experimental  inoculation  —  Action  on 
the  tissues  —  Mode  of  spread  —  Mallein  and  its  preparation —  Methods  of 
examination  —  Rhinoscleroma 275 

CHAPTER   XIII. 

ACTINOMYCOSIS   AND   ALLIED   DISEASES. 

Characters  of  the  actinomyces  —  Tissue  lesions  —  Distribution  of  lesions  — 
Cultivation  of  actinomyces  —  Varieties  of  actinomyces  and  allied  forms  — 
Experimental  inoculation  —  Methods  of  examination  and  diagnosis  — 
Madura  disease 287 

CHAPTER   XIV. 
ANTHRAX. 

Historical  summary  —  Bacillus  anthracis  —  Appearances  of  cultures  —  Biology 

—  Sporulation  —  Natural  anthrax  in  animals  —  Experimental  anthrax  — 
Anthrax  in  man  —  Pathology  —  Toxins  of  the  bacillus  anthracis  —  Mode 
of   spread    in    nature  —  Immunisation    against    anthrax  —  Methods   of 
examination 300 

CHAPTER   XV. 
TYPHOID  FEVER— BACILLI  ALLIED  TO  THE  TYPHOID  BACILLUS. 

Historical  summary  —  The  bacillus  typhosus  —  Morphological  characters  — 
Characters  of  cultures —  Bacillus  coli  communis  —  Reactions  of  B.  typhosus 
and  B.  coli — Bacillus  enteritidis  (Gaertner) — Paracolon  bacillus  —  Psit- 
tacosis bacillus  —  Pathological  changes  in  typhoid  fever  —  Suppuration  in 
typhoid  fever  —  Pathogenic  effects  produced  in  animals  —  The  toxic  pro- 
ducts of  B.  typhosus  —  Immunisation  of  animals  —  Relations  of  bacilli  to 
the  disease  —  Serum  diagnosis  —  Vaccination  against  typhoid  —  Methods 
of  examination  —  Bacillary  dysentery —  Bacillus  enteritidis  sporogenes  .  319 


xiv  CONTENTS. 

CHAPTER   XVI. 
DIPHTHERIA. 

PAGE 

Historical  —  General  facts  —  Bacillus  diphtheriae  —  Microscopical  characters  — 
Distribution  —  Cultivation  —  Inoculation  experiments  —  The  toxins  of 
diphtheria  —  Variations  in  virulence  of  bacilli  —  Summary  of  pathogenic 
action — Methods  of  diagnosis .  356 

CHAPTER   XVII. 

TETANUS. 

Introductory — Historical  —  Bacillus  tetani  —  Isolation  of  tetanus  bacillus  — 
Characters  of  cultures  —  Conditions  of  growth  —  Pathogenic  effects  — 
Experimental  inoculation  —  Tetanus  toxins  —  Antitetanic  serum  —  Methods 
of  examination  —  Malignant  oedema  —  Characters  of  bacillus  —  Methods 
of  diagnosis  —  Bacillus  botulinus  —  Quarter-evil  —  Bacillus  aerogenes 
capsulatus  —  Cultural  characters  .  .  .  .  .  .  .  .  376 

CHAPTER   XVIII. 
CHOLERA. 

Introductory  —  The  cholera  spirillum  —  Distribution  of  the  spirilla  —  Cultivation 

—  Powers  of  resistance  —  Experimental  inoculation  —  Toxins  of  cholera 
spirillum  —  Inoculation    of  human    subject  —  Immunity  —  Modes   of   dis- 
tinguishing  the    spirillum  —  General    summary  —  Spirilla   resembling    the 
cholera  organism  —  Methods   of  diagnosis  —  Metchnikoff 's    spirillum  — 
Finkler  and  Prior's  spirillum  —  Deneke's  spirillum 407 

CHAPTER  XIX. 

INFLUENZA,  PLAGUE,  RELAPSING  FEVER,  MALTA  FEVER, 
YELLOW  FEVER. 

Influenza  bacillus  —  Microscopical  characters  —  Cultivation  —  Distribution  — 
Experimental  inoculation — Methods  of  examination  —  Bacillus  of  plague 

—  Microscopical  characters  —  Cultivation  —  Anatomical  changes  produced 
and  distribution  of  bacilli  —  Experimental  inoculation  —  Paths  and  mode 
of  infection  —  Immunity — Relapsing  fever  —  Characters  of  the  spirillum 

—  Relations   to    the    disease  —  Malta    fever  —  Micrococcus   melitensis  — 
Relations  to  the  disease  —  Methods  of  diagnosis  —  Yellow  fever  —  Bacteria 

in  yellow  fever  —  The  mosquito  theory  of  infection 430 

CHAPTER   XX. 
IMMUNITY. 

Introductory  —  Acquired  immunity  —  Artificial  immunity  —  Varieties  —  Active 
immunity  —  Methods  of  production  — Attenuation  and  exaltation  of  viru- 
lence—  Passive  immunity  —  Action  of  the  serum — Antitoxic  serum  — 
Standardising  of  toxins  and  of  anti-sera  —  Nature  of  antitoxic  action  — 


/  CONTENTS.  xv 

PAGE 

Ehrlich's  theory  of  the  constitution  of  toxins  —  Antibacterial  serum  — 
Lysogenic  actions  —  Haemolytic  sera  —  Agglutination  —  Pfeiffer's  phe- 
nomenon —  Therapeutic  effects  of  anti-sera  —  Theories  as  to  acquired 
immunity  —  Ehrlich's  side-chain  theory  —  The  theory  of  phagocytosis  — 
Natural  immunity  —  Natural  bactericidal  powers  —  Natural  susceptibility 
to  toxins '  .  .  460 


APPENDIX    A. 
SMALLPOX  AND  VACCINATION. 

Jennerian  vaccination  —  Relationship  of  smallpox  and  cowpox  —  Bacteria  in 

smallpox  —  Protozoa  as  causative  agents  —  The  nature  of  vaccination        .     501 

APPENDIX   B. 
HYDROPHOBIA. 

Introductory  —  Pathology  —  The  hydrophobic  virus  —  Prophylaxis  —  Antirabic 

serum  —  Methods 508 

APPENDIX   C. 
MALARIAL  FEVER. 

Characters  of  the  malarial  parasite  —  Varieties  of  the  malarial  parasite  —  The 
cycle  of  the  malarial  parasite  in  man  —  The  cycle  in  the  mosquito  — 
Methods  of  examination „  .516 

APPENDIX   D. 
AMCEBIC  DYSENTERY. 

Amoebic  dysentery  —  Characters  of  the  amoeba  —  Distribution  of  the  amoeba  — 

Relations  to  disease — Methods  of  examination 529 

BIBLIOGRAPHY .         .     534 

INDEX     . .        .    553 


LIST   OF    ILLUSTRATIONS. 


FIG.  PAGE 

1.  Forms  of  bacteria 13 

2.  Hot-air  steriliser 29 

3.  Koch's  steam-steriliser 30 

4.  Arnold  steam-steriliser 30 

5.  Autoclave 31 

6.  Steriliser  for  blood  serum 32 

7.  Meat  press 34 

8.  Wire  funnel  for  supporting  paper  filter 39 

9.  Hot-water  funnel 40 

10.  Blood  serum  inspissator 44 

11.  Potato  jar 46 

12.  Cylinder  of  potato  cut  obliquely 47 

13.  Ehrlich's  tube  containing  piece  of  potato 47 

14.  Apparatus  for  filling  tubes 50 

15.  Tubes  of  media 50 

1 6.  Platinum  wires  in  glass  handles 51 

17.  Method  of  inoculating  solid  tubes 52 

1 8.  Rack  for  platinum  needles ,         .         .         -53 

19.  Petri's  capsule 53 

:2O.  Koch's  levelling  apparatus  for  use  in  preparing  plates          ....  55 

21.  Koch's  levelling  apparatus,  showing  transference  of  plate    ....  56 

22.  Esmarch's  tube  for  roll-culture 56 

23.  Apparatus  for  supplying  hydrogen  for  anaerobic  cultures    ....  59 

24.  Esmarch's  roll-tube  adapted  for  culture  containing  anaerobes     ...  60 

25.  Novy's  anaerobic  jar    ...........  6l 

26.  Bulloch's  apparatus  for  anaerobic  plate-cultures  .         .         .         .         .         .61 

27.  Flask  for  anaerobes  in  liquid  media     ........  63 

28.  Flask  arranged  for  culture  of  anaerobes  which  develop  gas          .         .         .64 

29.  Wright's  method  for  cultivation  of  anaerobes  in  solid  media       ...  65 
30,31.    Wright's  method  for  cultivation  of  anaerobes  in  fluid  media  .         .         .  65 

32.  Tubes  for  anaerobic  cultures  on  the  surface  of  solid  media           ...  66 

33.  Harris'  method  of  making  collodion  sacs     .         .         .         .         .         .         -67 

34.  Slide  for  hanging-drop  cultures 68 

35.  Graham  Brown's  chamber  for  anaerobic  hanging-drops       ....  69 

36.  Sternberg's  bulb  adapted  for  thermal  death-point  test         .         .         .         •  71 

37.  Apparatus  for  counting  colonies  .........  72 

38.  Geissler's  vacuum-pump  for  filtering  cultures       ......  75 

39.  Chamberland's  candle  and  flask  arranged  for  filtration         ....  76 

40.  Chamberland's  bougie,  with  lamp  funnel     .......  7^ 

41.  Bougie  inserted  through  rubber  stopper 76 

xvii 


xviii  LIST   OF   ILLUSTRATIONS. 

FIG.  PAGE 

42.  Muencke's  modification  of  Chamber-land's  filter 77 

43.  Flask  for  filtering  large  quantities  of  fluid 77 

44.  Tubes  for  demonstrating  gas-formation  by  bacteria 79 

45.  Hill's  modification  of  Smith's  fermentation  tube 80 

46.  Geryk's  air-pump  for  drying  in  vacua  .         .         .         .         .         .         .81 

47.  Reichert's  gas  regulator 83 

48.  Hearson's  incubator  for  use  at  37°  C 84 

49.  Cornet's  forceps  for  holding  cover-glasses 89 

50.  Stewart's  cover-glass  forceps 89 

51.  Needle  for  manipulating  paraffin  sections 94 

52.  Syphon  wash-bottle .98 

53.  Tubes  used  in  testing  agglutinating  and  sedimenting  properties  of  serum    .     ill 

54.  Test-tube  and  pipette  for  obtaining  fluids  containing  bacteria      .         .         .     113 

55.  Apparatus  for  holding  mouse  preparatory  to  subcutaneous  inoculation        .     118 

56.  Hollow  needle  for  intraperitoneal  inoculations .119 

57.  Hesse's  tube 124 

58.  Petri's  sand  filter .125 

59.  Sedgwick-Tucker  aerobioscope -125 

60.  Apparatus  for  collecting  water  samples 133 

61.  Forms  of  fungi 150 

62.  Blastomyces  dermatitidis,  showing  organism  with  doubly-contoured  mem- 

brane,     x  1000 153 

63.  Blastomyces  dermatitidis,  showing  budding  form  of  organism.      X  1000      .      153 

64.  Blastomyces  dermatitidis,  sporulating  form  in  abscess  cavity        .         .         •     154 

65.  Staphylococcus  pyogenes  aureus,  young  culture  on  agar.      X  1000      .         .183 

66.  Two  stab-cultures  of  Staphylococcus  pyogenes  aureus  in  gelatin  .         .     183 

67.  Streptococcus  pyogenes,  young  culture  on  agar.      x  1000  .         .         .         .185 

68.  Culture  of  the  streptococcus  pyogenes  on  an  agar  plate       .         .         .         .185 

69.  Bacillus  pyocyaneus,  young  culture  on  agar.      x  1000          .         .         .         .186 

70.  Micrococcus  tetragenus.      x  1000 .187 

71.  Film  preparation  of  exudation  from  case  of  meningitis,  showing  the  diplo- 

cocci  within  leucocytes .188 

72.  Streptococci  in  acute  suppuration,      x  1000         .         .         .         .         .         .     193 

73.  Minute  focus  of  commencing  suppuration  in  brain.      X  50  .         .          .         .     195 

74.  Secondary  infection    of  a   glomerulus  of  kidney  by  the   Staphylococcus 

aureus.      x  300 196 

75.  Section  of  a  vegetation  in  ulcerative  endocarditis 198 

76.  Film  preparation  of  conjunctival  secretion,      x  1000 201 

77.  Film  preparation  of  pneumonic  sputum,      x  1000 208 

78.  Friedlander's    pneumobacillus,    from    exudate    in    a    case    of    pneumonia. 

X  1000 209 

79.  Fraenkel's  pneumococcus  in  serous  exudation,      x  1000      ....     209 

80.  Stroke-culture  of  Fraenkel's  pneumococcus  on  blood  agar  .         .         .         .210 

81.  Fraenkel's  pneumococcus  from  a  pure  culture  on  blood  agar  of  twenty-four 

hours' growth,      x  1000 211 

82.  Stab-culture  of  Friedlander's  pneumobacillus 212 

83.  Friedlander's  pneumobacillus  from  a  young  culture  on  agar.      x  1000          .  212 

84.  Capsulated   pneumococci    in   blood    taken    from    the    heart    of  a    rabbit. 

X  1000 215 


LIST   OF   ILLUSTRATIONS.  xix 

FIG.  PAGE 

85.  Portion  of  film  of  gonorrhoeal  pus.      x  1000 224 

86.  Gonococci,  from  a  pure  culture  on  blood  agar.      x  1000   ....  225 

87.  Tubercle  bacilli,  from  a  pure  culture,  on  glycerin  agar.      x  1000         .         .  239 

88.  Tubercle  bacilli  in  phthisical  sputum,      x  1000          .         .         .         .         .  239 

89.  Cultures  of  tubercle  bacilli  on  glycerin  agar 242 

90.  Tubercle  bacilli  in  section  of  human  lung  in  acute  phthisis.      X  1000         .  247 

91.  Tubercle  bacilli  in  giant-cells.      X  1000 248 

92.  Tubercle  bacilli  in  urine.      X  1000 248 

/93.    Moeller's  Timothy-grass  bacillus,      x  1000 255 

94.  Cultures  of  acid-fast  bacilli  grown  at  room  temperature      ....  256 

95.  Smegma  bacilli.      X  1000 256 

96.  Section  through  leprous  skin,  showing  the  masses  of  cellular  granulation 

tissue  in  the  cutis.      X  80 268 

97.  Superficial  part  of  leprous  skin  ;   the  cells  of  the  granulation  tissue  appear 

as  dark  patches.      X  500 270 

98.  High-power  view  of  portion  of  leprous  nodule.      X  uoo  ....  271 

99.  Glanders  bacilli  amongst  broken-down  cells,      x  1000        ....  277 

100.  Glanders  bacilli,  from  a  pure  culture.      X  1000 278 

101.  Actinomycosis  of  human  liver.      X  500 289 

102.  Actinomyces  in  human  kidney.      X  500 290 

103.  Colonies  of  actinomyces.      X  60 291 

104.  Cultures  of  the  actinomyces  on  glycerin  agar 294 

105.  Actinomyces,  from  a  culture  on  glycerin  agar.      x  1000     ....  294 

1 06.  Streptothrix  madurse.      X  1000           ........  298 

107.  Surface  colony  of  the  anthrax  bacillus.      X  30 302 

108.  Anthrax  bacilli,  arranged  in  chains.      X  1000 302 

109.  Stab-culture  of  the  anthrax  bacillus 303 

no.    Anthrax  bacilli  containing  spores.      X  1000 305 

in.    Scraping  from  spleen  of  guinea-pig  dead  of  anthrax,      x  1000  .         .         .  307 

112.  Portion  of  kidney  of  a  guinea-pig  dead  of  anthrax.      X  300       .         .         .  309 

113.  Specially  large  clump  of  typhoid  bacilli  in  a  spleen.      X  500      .         .         .  320 

114.  Typhoid  bacilli.      X  1000 321 

115.  Typhoid  bacilli  from  a  young  culture  on  agar.      x  1000    ....  322 

1 1 6.  Stab  and  stroke  cultures  of  typhoid  bacillus  and  bacillus  coli     .         .         .  323 

117.  Colonies  of  typhoid  bacillus.      X  15  .         .         .         .         .         .         .         .  323 

118.  Bacillus  coli  communis.      X  1000 325 

118  A.   Bacillus  dysenteriae,  from  an  agar  culture  48  hours  old.      X  1000  .         .  350 

119.  Film  preparation  from  diphtheria  membrane,      x  1000     ....  358 

1 20.  Section  through  a  diphtheritic  membrane,  showing  diphtheria  bacilli  in 

clumps,      x  1000 359 

121.  Cultures  of  the  diphtheria  bacillus  on  an  agar  plate 361 

122.  Diphtheria  bacilli,  from  a  twenty-four  hours'  culture  on  agar.      X  1000    .  362 

123.  Diphtheria  bacilli  of  larger  size  than  in  previous  figure.      X  1000       .         .  362 

124.  Involution  forms  of  the  diphtheria  bacillus,      x  1000         ....  362 

125.  Pseudo-diphtheria  bacillus  (Hofmann's).     Young  agar   culture.      X  1000  371 

126.  Film  preparation  of  discharge  from  wound  in  a  case  of  tetanus.      X  1000  377 

127.  Tetanus  bacilli,  showing  flagella.     x  1000 •  378 

128.  Spiral   composed  of   numerous   twisted   flagella   of  the   tetanus  bacillus. 

X  1000 378 


xx  LIST   OF   ILLUSTRATIONS. 

FIG.  PAGE. 

129.  Tetanus  bacilli,  some  of  which  possess  spores,     x  1000    ....  379 

130.  Bipolar  spore  formation  in  glucose  agar  culture  of  B.  tetani.      X  1000      .  379 

131.  Stab-culture  of  the  tetanus  bacillus  (Kitasato) 381 

132.  Film  preparation  in  a  case  of  malignant  oedema  in  human  subject.      X  1000  394 

133.  Bacillus  of  malignant  oedema.      X  1000 395 

134.  Stab-cultures  in  agar 396 

135.  Bacillus  of  quarter-evil,  showing  spores,      x  1000 401 

136.  Bacillus  aerogenes  capsulatus 403 

137.  B.  aerogenes  capsulatus,  showing  coagulation  of  casein     ....  405 

138.  Cholera  spirilla,      x  1000 409 

139.  Cholera  spirilla  stained  to  show  the  terminal  flagella.      x  1000          .         .  409 

140.  Cholera  spirilla  from  an  old  agar  culture,      x  1000    .....  409 

141.  Puncture  culture  of  the  cholera  spirillum 411 

142.  Colonies  of  the  cholera  spirillum  in  a  gelatin  plate 412 

143.  Metchnikoff  s  spirillum,      x  1000 427 

144.  Puncture  cultures  in  peptone  gelatin 428 

145.  Finkler  and  Prior's  spirillum  from  an  agar  culture,      x  1000     .         .         .  429 

146.  Influenza  bacilli  from  a  culture  on  blood  agar.      x  1000   ....  430 

147.  Film  preparation   from  a  plague   bubo,  showing  enormous  numbers  of 

bacilli.      X  1000 436 

148.  Bacillus  of  plague  from  a  young  culture  on  agar.      x  1000        .         .         .  436 

149.  Bacillus  of  plague  in  chains.      X  1000 437 

150.  Culture  of  the  bacillus  of  plague  on  4  per  cent  salt  agar.      x  1000    .         .  437 

151.  Section  of  a  human  lymphatic  gland  in  plague,      x  50      .         .         .         .  439 

152.  Film  preparation  of  spleen  of  rat  after  inoculation  with  the  bacillus  of 

plague,      x  1000 441 

153.  Spirilla  of  relapsing  fever  in  human  blood.      X  about  1000        .         .         .  448 

154.  Micrococcus  melitensis,  from  a  two  days' culture  on  agar  at  37°  C.      X  1000  453 

155.  Bacillus .icteroides,  from  a  young  culture  on  agar  (Sanarelli).      X  looo     .  456 

156.  Culture  of  Bacillus  icteroides  on  agar.     Natural  size  .         .         .         .  457 

157-162.   Various  phases  of  the  benign  tertian  parasite 518 

163-168.    Exemplifying  phases  of  the  malignant  parasite 519 

169.  Amoebae  of  dysentery .  530 

170.  Section  of  wall  of  liver  abscess,  showing  an  amoeba  of  spherical  form  with 

vacuolated  protoplasm,     x  1000 532 


MANUAL   OF   BACTERIOLOGY 


MANUAL  OF  BACTERIOLOGY. 

CHAPTER   I. 
GENERAL   MORPHOLOGY   AND   BIOLOGt. 

Introductory.  —  At  the  bottom  of  the  scale  of  living  things 
there  exists  a  group  of  organisms  to  which  the  name  of  bacteria 
is  usually  applied.  These  are  apparently  of  very  simple  struc- 
ture and  may  be  subdivided  into  two  sub-groups,  a  lower  and 
simpler  and  a  higher  and  better  developed. 

The  lower  forms  are  the  more  numerous,  and  consist  of 
minute  unicellular  masses  of  protoplasm  devoid  of  chlorophyll, 
which  multiply  by  simple  fission.  Some  are  motile,  others  non- 
motile.  Their  minuteness  may  be  judged  of  by  the  fact  that  in 
one  direction  at  least  they  usually  do  not  measure  more  than 
i  fji  (25^00"  mcn)-  These  forms  can  be  classified  according  to 
their  shapes  into  three  main  groups — (i)  A  group  in  which  the 
shape  is  globular.  The  members  of  this  are  called  cocci.  (2)  A 
group  in  which  the  shape  is  that  of  a  straight  rod  —  the  propor- 
tion of  the  length  to  the  breadth  of  the  rod  varying  greatly 
among  the  different  members.  These  are  called  bacilli.  (3)  A 
group  in  which  the  shape  is  that  of  a  curved  or  spiral  rod. 
These  are  called  spirilla.  The  full  description  of  the  characters 
of  these  groups  will  be  more  conveniently  taken  later  (p.  12). 
In  some  cases,  especially  among  the  bacilli,  there  may  occur 
under  certain  circumstances  changes  in  the  protoplasm  whereby 
a  resting  stage  or  spore  is  formed. 

The  higher  forms  show  advance  on  the  lower  along  two 
lines,  (i)  On  the  one  hand  they  consist  of  filaments  made  up 
of  simple  elements  such  as  occur  in  the  lower  forms.  These 
filaments  may  be  more  or  less  septate,  may  be  provided  with  a 
sheath,  and  may  show  branching  either  true  or  false.  The 


GENERAL   MORPHOLOGY  AND    BIOLOGY. 

minute  structure  of  the  elements  comprising  these  filaments 
is  analogous  to  that  of  the  lower  forms.  Their  size,  however,  is 
often  somewhat  greater.  The  lower  forms  sometimes  occur  in 
filaments,  but  here  every  member  of  the  filament  is  independent, 
while  in  the  higher  forms  there  seems  to  be  a  certain  interde- 
pendence among  the  individual  elements.  For  instance,  growth 
may  occur  only  at  one  end  of  a  filament,  the  other  forming  an 
attachment  to  some  fixed  object.  (2)  The  higher  forms,  more- 
over, present  this  further  development  that  in  certain  cases  some 
of  the  elements  may  be  set  apart  for  the  reproduction  of  new 
individuals. 

Terminology.  —  The  term  bacterium  of  course  in  strictness 
only  refers  to  the  rod-shaped  varieties  of  the  group,  but  as  it  has 
given  the  name  bacteriology  to  the  science  which  deals  with  the 
whole  group,  it  is  convenient  to  apply  it  to  all  the  members  of 
the  latter,  and  to  reserve  the  term  bacillus  for  the  rod-shaped 
varieties.  Other  general  words,  such  as  germ,  microbe,  micro- 
organism, are  often  used  as  synonymous  with  bacterium,  though, 
strictly,  they  include  the  smallest  organisms  of  the  animal  king- 
dom. 

While  no  living  organisms  lower  than  the  bacteria  are  known 
(though  the  occurrence  of  such  is  now  suspected),  the  upper 
limits  of  the  group  are  difficult  to  define,  and  it  is  further  impos- 
sible in  the  present  state  of  our  knowledge  to  give  other  than  a 
provisional  classification  of  the  forms  which  all  recognise  to  be 
bacteria.  The  division  into  lower  and  higher  forms,  however, 
is  fairly  well  marked,  and  we  shall  therefore  refer  to  the  former 
as  the  lower  bacteria,  and  to  the  latter  as  the  higher  bacteria. 

Morphological  Relations.  — The  relations  of  the  bacteria  to  the  animal 
kingdom  on  the  one  hand  and  to  the  vegetable  on  the  other  constitute  a  some- 
what difficult  question.  It  is  best  to  think  of  there  being  a  group  of  small,, 
unicellular  organisms,  which  may  represent  the  most  primitive  forms  of  life 
before  differentiation  into  animal  and  vegetable  types  had  occurred.  This 
would  include  the  flagellata  and  infusoria,  the  myxomycetes,  the  lower  algae, 
and  the  bacteria.  To  the  lower  algae  the  bacteria  possess  many  similarities. 
These  algae  are  unicellular  masses  of  protoplasm,  having  generally  the  same 
shapes  as  the  bacteria,  and  largely  multiply  by  fission.  Endogenous  sporula- 
tion  however,  does  not  occur,  nor  is  motility  associated  with  the  possession 
of  flagella.  Also  their  protoplasm  differs  from  that  of  the  bacteria  in  contain- 
ing chlorophyll  and  another  blue-green  pigment  called  phycocyan.  From  the 
morphological  resemblances,  however,  between  these  algae  and  the  bacteria, 
and  from  the  fact  that  fission  plays  a  predominant  part  in  the  multiplication 


STRUCTURE  OF  THE  BACTERIAL  CELL.        3 

of  both,  they  have  been  grouped  together  in. one  class  as  the  Schizophyta  or 
splitting  plants  (German,  Spaltpflanzen) .  And  of  the  two  divisions  forming 
these  Schizophyta  the  splitting  algae  are  denominated  the  schizophyceae  (Ger- 
man, Spaltalgen),  while  the  bacteria  or  splitting  fungi  are  called  the  schizo- 
mycetes  (German,  Spaltpilzen).  The  bacteria  are,  therefore,  often  spoken*  of 
as  the  schizomycetes.  Certain  bacteria  which  have  been  described  as  con- 
taining chlorophyll  ought  probably  to  be  grouped  among  the  schizophyceae. 

GENERAL  MORPHOLOGY  OF  THE  BACTERIA. 

The  Structure  of  the  Bacterial  Cell.  —  On  account  of  the 
minuteness  of  bacteria  the  investigation  of  their  structure  is 
attended  with  great  difficulty.  When  examined  under  the 
microscope,  in  their  natural  condition,  e.g.  in  water,  they 
appear  merely  as  colourless  refractile  bodies  of  the  different 
shapes  named.  Spore  formation  and  motility,  when  these  exist, 
can  also  be  observed,  but  little  else  can  be  made  out.  For 
their  proper  investigation  advantage  is  always  taken  of  the  fact 
of  their  affinities  for  various  dyes,  especially  those  which  are 
usually  chosen  as  good  stains  for  the  nuclei  of  animal  cells. 
Certain  points  have  thus  been  determined.  The  bacterial  cell 
consists  of  a  sharply  contoured  mass  of  protoplasm  which 
reacts  to,  especially  basic,  aniline  dyes  like  the  nucleus  of  an 
animal  cell  —  though  from  this  fact  we  cannot  deduce  that  the 
two  are  identical  in  "composition.  A  healthy  bacterium  when 
thus  stained  presents  the  appearance  of  a  finely  granular  or 
almost  homogeneous  structure.  The  protoplasm  is  surrounded 
by  an  envelope  which  can  in  some  cases  be  demonstrated  by 
overstaining  a  specimen  with  a  strong  aniline  dye,  when  it  will 
appear  as  a  halo  round  the  bacterium.  This  envelope  may  some- 
times be  seen  to  be  of  considerable  thickness.  Its  innermost 
layer  is  probably  of  a  denser  consistence,  and  sharply  contours 
the  contained  protoplasm,  giving  the  latter  the  appearance  of 
being  surrounded  by  a  membrane.  It  is  only,  however,  in  some 
of  the  higher  forms  that  a  true  membrane  occurs.  Sometimes 
the  outer  margin  of  the  envelope  is  sharply  defined,  in  which 
case  the  bacterium  appears  to  have  a  distinct  capsule,  and  is 
known  as  a  capsulated  bacterium  (vide  Fig.  I,  No.  4;  and 
Fig.  77).  The  cohesion  of  bacteria  into  masses  depends  largely 
on  the  character  of  the  envelope.  If  the  latter  is  glutinous, 
then  a  large  mass  of  the  same  species  may  occur,  formed  of  in- 
dividual bacteria  embedded  in  what  appears  to  be  a  mass  of  jelly. 


4  GENERAL   MORPHOLOGY  AND   BIOLOGY. 

When  this  occurs,  it  is  known  as  a  zoogloea  mass.  On  the  other 
hand,  if  the  envelope  has  not  this  cohesive  property  the  sepa- 
ration of  individuals  may  easily  take  place,  especially  in  a  fluid 
medium  in  which  they  may  float  entirely  free  from  one  another. 
Many  of  the  higher  bacteria  possess  a  sheath  which  has  a  much 
more  definite  structure  than  is  found  among  the  lower  forms. 
It  resists  external  influences,  possesses  elasticity,  and  serves  to 
bind  the  elements  of  the  organism  together. 

Reproduction  among  the  Lower  Bacteria. — When  a  bacterial 
cell  is  placed  in  favourable  surroundings  it  multiplies ;  as  has 
been  said,  this,  in  the  great  majority  of  cases,  takes  place  by 
simple  fission.  In  the  process  a  constriction  appears  in  the 
middle  and  a  transverse  unstained  line  develops  across  the  proto- 
plasm at  that  point.  The  process  goes  on  till  two  individuals 
can  be  recognised,  which  may  remain  for  a  time  attached  to  one 
another,  or  become  separate,  according  to  the  character  of  the 
envelope,  as  already  explained.  In  most  bacteria  growth  and 
multiplication  go  on  with  great  rapidity.  A  bacterium  may 
reach  maturity  and  divide  in  from  twenty  minutes  to  half  an 
hour.  If  division  takes  place  only  every  hour,  from  one  indi- 
vidual after  twenty-four  hours  17,000,000  similar  individuals  will 
be  produced.  As  shown  by  the  results  of  artificial  cultivation, 
others,  such  as  the  tubercle  bacillus,  multiply  much  more  slowly. 
Sometimes  division  proceeds  so  rapidly  that  the  young  indi- 
viduals do  not  reach  the  adult  size  before  multiplication  again 
occurs.  This  may  give  rise  to  anomalous  appearances.  When 
bacteria  are  placed  in  unfavourable  conditions  as  regards  food, 
etc.,  growth  and  multiplication  take  place  with  difficulty.  In 
the  great  majority  of  cases  this  is  evidenced  by  changes  in  the 
appearance  of  the  protoplasm.  Instead  of  its  maintaining  the 
regularity  of  shape  seen  in  healthy  bacteria,  various  aberrant 
appearances  are  presented.  This  occurs  especially  in  the  rod- 
shaped  varieties,  where  flask-shaped  or  dumb-bell-shaped  indi- 
viduals may  be  seen.  The  regularity  in  structure  and  size  is 
quite  lost.  The  appearance  of  the  protoplasm  also  is  often 
altered.  Instead  of,  as  formerly,  staining  well,  it  does  not  stain 
readily,  and  may  have  a  uniformly  pale,  homogeneous  appear- 
ance, while  in  an  old  culture  only  a  small  proportion  of  the 
bacteria  may  stain  at  all.  Sometimes,  on  the  other  hand,  a 
degenerated  bacterium  contains  intensely  stained  granules  or 


SPORE   FORMATION.  5 

globules  which  may  be  of  large  size.  Such  aberrant  and  degen- 
erate appearances  are  referred  to  as  involution  forms.  That 
these  forms  really  betoken  degenerative  changes  is  shown  by 
the  fact  that,  on  their  being  again  transferred  to  favourable 
conditions,  only  slight  growth  at  first  takes  place.  Many  indi- 
viduals have  undoubtedly  died,  and  the  remainder  which  live  and 
develop  into  typical  forms  may  sometimes  have  lost  some  of  their 
properties. 

Reproduction  among  the  Higher  Bacteria. —  Most  of  the  higher  bacteria 
consist  of  thread-like  structures  more  or  less  septate,  and  often  surrounded  by 
a  sheath.  The  organism  is  frequently  attached  at  one  end  to  some  object  or 
to  another  individual.  It  grows  to  a  certain  length,  and  then  at  the  free  end 
certain  cells  called  gonidia  are  cast  off,  from  which  new  individuals  are  formed. 
These  gonidia  may  be  formed  by  a  division  taking  place  in  the  terminal  ele- 
ment of  the  filament,  such  as  has  occurred  in  the  growth  of  the  latter.  In 
some  cases,  however,  division  takes  place  in  three  dimensions  of  space.  The 
gonidia  have  a  free  ^existence  for  a  certain  time  before  becoming  attached, 
and  in  this  stage  are  sometimes  motile.  They  are  usually  rod-like  in  shape, 
sometimes  pyriform.  They  do  not  possess  any  special  powers  of  resistance. 

Spore  Formation.  —  In  certain  species  of  the  lower  bacteria, 
under  certain  circumstances,  changes  take  place  in  the  proto- 
plasm which  result  in  the  formation  of  bodies  called  spores,  to 
which  the  vital  activities  of  the  original  bacteria  are  transferred. 
Spore  formation  occurs  chiefly  among  the  bacilli  and  in  some 
spirilla.  Its  commencement  in  a  bacterium  is  indicated  by  the 
appearance  in  the  protoplasm  of  a  minute  highly  refractile 
granule  (or  by  a  number  of  minute  highly  refractile  granules 
scattered  about  throughout  the  protoplasm  which  gradually 
coalesce)  unstained  by  the  ordinary  methods.  This  increases  in 
size,  and  assumes  a  round,  oval,  or  short  rod-shaped  form,  always 
shorter  but  often  broader  than  the  original  bacterium.  In  the 
process  of  spore  formation  the  rest  of  the  bacterial  protoplasm 
may  remain  unchanged  in  appearance  and  staining  power  for  a 
considerable  time  (e.g.  B.  tetani\  or,  on  the  other  hand,  it  may 
soon  lose  its  power  of  staining  and  ultimately  disappear,  leaving 
the  spore  in  the  remains  of  the  envelope  (e.g.  B.  anthracis). 
This  method  of  spore  formation  is  called  endogenous.  Bacterial 
spores  are  always  non-motile.  The  spore  may  appear  in  the 
centre  of  the  bacterium,  or  it  may  be  at  one  extremity,  or  a 
short  distance  from  one  extremity  (Fig.  I,  No.  1 1).  In  structure 
the  spore  consists  of  a  mass  of  protoplasm  surrounded  by  a  dense 


6  GENERAL   MORPHOLOGY  AND   BIOLOGY. 

membrane.  This  can  be  demonstrated  by  methods  which  will 
be  described,  the  underlying  principle  of  which  is  the  prolonged 
application  of  a  powerful  stain.  The  membrane  is  supposed 
to  confer  on  the  spore  its  characteristic  feature,  namely,  great 
capacity  of  resistance  to  external  influences  such  as  heat  or 
noxious  chemicals.  Koch,  for  instance,  in  one  series  of  experi- 
ments, found  that  while  the  bacillus  anthracis  in  the  unspored 
form  was  killed  by  a  two  minutes'  exposure  to  I  per  cent  carbolic 
acid,  spores  of  the  same  organism  resisted  an  exposure  of  from 
one  to  fifteen  days. 

When  a  spore  is  placed  in  suitable  surroundings  for  growth  it 
again  assumes  the  original  bacillary  or  spiral  form.  The  capsule 
dehisces  either  longitudinally,  or  terminally,  or  transversely.  In 
the  last  case  the  dehiscence  may  be  partial,  and  the  new  indi- 
vidual may  remain  for  a  time  attached  by  its  ends  to  the  hinged 
spore-case,  or  the  dehiscence  may  be  complete  and  the  bacillus 
grow  with  a  cap  at  each  end  consisting  of  half  the  spore-case. 
Sometimes  the  spore-case  does  not  dehisce,  but  is  simply  absorbed 
by  the  developing  bacterium. 

It  is  important  to  note  that  in  the  bacteria  spore  formation 
is  rarely,  if  ever,  to  be  considered  as  a  method  of  multiplication. 
In  at  least  the  great  majority  of  cases  only  one  spore  is  formed 
from  one  bacterium,  and  only  one  bacterium  in  the  first  instance 
from  one  spore.  Sporulation  is  to  be  looked  upon  as  a  resting 
stage  of  a  bacterium,  and  is  to  be  contrasted  with  the  stage 
when  active  multiplication  takes  place.  The  latter  is  usually 
referred  to  as  the  vegetative  stage  of  the  bacterium.  Regarding 
the  signification  of  spore  formation  in  bacteria  there  has  been 
some  difference  of  opinion.  According  to  one  view  it  may  be 
regarded  as  representing  the  highest  stage  in  the  vital  activity 
of  a  bacterium.  There  is  thus  an  alternation  between  the 
vegetative  and  spore  stage,  the  occurrence  of  the  latter  being 
necessary  to  the  maintenance  of  the  species  in  its  greatest 
vitality.  Such  a  rejuvenescence,  as  it  were,  through  sporulation, 
is  known  in  many  algae.  In  support  of  this  view  there  are 
certain  facts.  In  many  cases,  for  instance,  spore  formation 
only  occurs  at  temperatures  specially  favourable  for  growth  and 
multiplication.  There  is  often  a  temperature  below  which, 
while  vegetative  growth  still  takes  place,  sporulation  will  not 
occur,  and  in  the  case  of  B.  anthracis,  if  the  organism  be  kept 


QUESTION   OF   ARTHROSPOROUS    BACTERIA.  7 

at  a  temperature  above  the  limit  at  which  it  grows  best,  not 
only  are  no  spores  formed,  but  the  species  may  lose  the  power 
of  sporulation.  Furthermore,  in  the  case  of  bacteria  preferring 
the  presence  of  oxygen  for  their  growth,  an  abundant  supply  of 
this  gas  may  favour  sporulation.  Most  bacteriologists  are, 
however,  of  opinion  that  when  a  bacterium  forms  a  spore,  it 
only  does  so  when  its  surroundings,  especially  its  food  supply, 
become  unfavourable  for  vegetative  growth ;  it  then  remains  in 
this  condition  until  it  is  placed  in  more  suitable  surroundings. 
Such  an  occurrence  would  be  analogous  to  what  takes  place  un- 
der similar  conditions  in  many  of  the  protozoa.  Often  sporu- 
lation can  be  prevented  from  taking  place  for  an  indefinite  time 
if  a  bacterium  is  constantly  supplied  with  fresh  food  (the  other 
conditions  of  life  being  equal).  The  presence  of  substances 
excreted  by  the  bacteria  themselves  plays,  however/  a  more 
important  part  in  making  the  surroundings  unfavourable  than 
the  mere  exhaustion  of  the  food  supply.  A  living  spore  will 
always  develop  into  a  vegetative  form  if  placed  in  a  fresh  food 
supply.  With  regard  to  the  rapid  formation  of  spores  when  the 
conditions  are  favourable  for  vegetative  growth,  it  must  be 
borne  in  mind  that  in  such  circumstances  the  conditions  may 
really  very  quickly  become  unfavourable  for  a  continuance  of 
growth,  since  not  only  will  the  food  supply  around  the  growing 
bacteria  be  rapidly  exhausted,  but  the  excretion  of  effete  and 
inimical  matters  will  be  all  the  more  rapid. 

We  must  note  that  the  usually  applied  tests  of  a  body  de- 
veloped within  a  bacterium  being  a  spore  are  (i)  its  staining 
reaction,  namely,  resistance  to  ordinary  staining  fluids,  but 
capacity  of  being  stained  by  the  special  methods  devised  for 
the  purpose  (vide  p.  106) ;  (2)  the  fact  that  the  bacterium 
containing  the  spore  has  higher  powers  of  resistance  against 
inimical  conditions  than  a  vegetative  form.  It  is  important 
to  bear  these  tests  in  mind,  as,  in  some  of  the  smaller  bacteria 
especially,  it  is  very  difficult  to  say  whether  they  spore  or  not. 
There  may  appear  in  such  organisms  small  unstained  spots  the 
significance  of  which  it  is  very  difficult  to  determine. 

The  Question  of  Arthrosporous  Bacteria.  —  It  is  stated  by  Hueppe  that 
among  certain  organisms,  e.g.  some  streptococci,  certain  individuals  may, 
without  endogenous  sporulation,  take  on  a  resting  stage.  These  become 
swollen,  stain  well  with  ordinary  stains,  and  they  are  stated  to  have  higher 


8  GENERAL   MORPHOLOGY   AND    BIOLOGY. 

power  of  resistance  than  the  other  forms  ;  further,  when  vegetative  life  again 
occurs  it  is  from  them  that  multiplication  is  said  to  take  place.  From  the 
fact  that  there  is  no  new  formation  within  the  protoplasm,  but  that  it  is  the 
whole  of  the  latter  which  participates  in  the  change,  these  individuals  have 
been  called  arthrospores.  The  existence  of  such  special  individuals  amongst 
the  lower  bacteria  is  extremely  problematical.  They  have  no  distinct  capsule, 
and  they  present  no  special  staining  reactions,  nor  any  microscopic  features. 
by  which  they  can  be  certainly  recognised,  while  their  alleged  increased 
powers  of  resistance  are  very  doubtful.  All  the  phenomena  noted  can  be 
explained  by  the  undoubted  fact  that  in  an  ordinary  growth  there  is  very 
great  variation  among  the  individual  organisms  in  their  powers  of  resistance 
to  external  conditions. 

Motility.  —  As  has  been  stated,  many  bacteria  are  motile 
Motility  can  be  studied  by  means  of  hanging-drop  preparations 
(vide  p.  68).  The  movements  are  of  a  darting,  rolling,  or 
vibratile  character.  The  degree  of  motility  depends  on  the 
temperature,  on  the  age  of  the  growth,  and  on  the  medium  in 
which  the  bacteria  are.  Sometimes  the  movements  are  most 
active  just  after  the  cell  has  multiplied,  sometimes  it  goes  on 
all  through  the  life  of  the  bacterium,  sometimes  it  ceases  when 
sporulation  is  about  to  occur.  Motility  is  associated  with  the 
possession  of  fine  wavy  thread-like  appendages  called  flagella, 
which  for  their  demonstration  require  the  application  of  special 
staining  methods  (vide  Fig.  I,  No.  12;  and  Fig.  115).  They 
have  been  shown  to  occur  in  many  bacilli  and  spirilla,  but  only 
in  a  few  species  of  cocci.  They  vary  in  length,  but  may  be 
several  times  the  length  of  the  bacterium,  and  may  be  at  one 
or  both  extremities  or  all  round.  When  terminal  they  may 
occur  singly  or  there  may  be  several.  The  nature  of  these 
flagella  has  been  much  disputed.  Some  have  held  that,  unlike 
what  occurs  in  many  algae,  they  are  not  actual  prolongations 
of  the  bacterial  protoplasm,  but  merely  appendages  of  the  en- 
velope, and  have  doubted  whether  they  are  really  organs  of 
locomotion.  There  is  now,  however,  little  doubt  that  they  be- 
long to  the  protoplasm.  By  appropriate  means  the  central  parts 
of  the  latter  can  be  made  to  shrink  away  from  the  peripheral 
(vide  infra,  "  plasmolysis  ").  In  such  a  case  movement  goes  on 
as  before,  and  in  stained  preparations  the  flagella  can  be  seen 
to  be  attached  to  the  peripheral  zone.  It  is  to  be  noted  that 
flagella  have  never  been  demonstrated  in  non-motile  bacteria, 
while,  on  the  other  hand,  they  have  been  observed  in  nearly 


MOTILITY.  9 

all  motile  forms.  There  is  little  doubt,  however,  that  all  cases 
of  motility  among  the  bacteria  are  not  dependent  on  the  posses- 
sion of  flagella,  for  in  some  of  the  special  spiral  forms,  and  in 
most  of  the  higher  bacteria,  motility  is  probably  due  to  contrac- 
tility of  the  protoplasm  itself. 

The  Minuter  Structure  of  the  Bacterial  Protoplasm.  —  Many  attempts  have 
been  made  to  obtain  deeper  information  as  to  the  structure  of  the  bacterial 
cell,  and  especially  as  to  its  behaviour  in  division.  These  have  largely  turned 
on  the  interpretation  to  be  put  on  certain  appearances  which  have  been  ob- 
served. These  appearances  are  of  two  kinds.  First,  under  certain  circum- 
stances irregular,  deeply  stained  granules  are  observed  in  the  protoplasm, 
often,  when  they  occur  in  a  bacillus,  giving  the  latter  the  appearance  of  a 
short  chain  of  cocci.  They  are  often  called  metachromatic  granules  (vide 
Fig.  i,  No.  1 6)  from  the  fact  that  by  appropriate  procedure  they  can  be 
stained  with  one  dye,  and  the  protoplasm  in  which  they  lie  with  another; 
sometimes,  when  a  single  stain  is  used,  such  as  methylene-blue,  they  assume 
a  slightly  different  tint  from  the  protoplasm. 

For  the  demonstration  of  the  metachromatic  granules  two  methods  have 
been  advanced.  Ernst  recommends  that  a  few  drops  of  Loffler's  methylene- 
blue  (vide  p.  100)  be  placed  on  a  cover-glass  preparation  and  the  latter  passed 
backwards  and  forwards  over  a  Bunsen  flame  for  half  a  minute  after  steam 
begins  to  rise.  The  preparation  is  then  washed  in  water  and  counter-stained 
for  one  or  two  minutes  in  watery  Bismarck-brown.  The  granules  are  here 
stained  blue,  the  protoplasm  brown.  Neisser  stains  a  similar  preparation  in 
warm  carbol-fuchsin,  washes  with  I  per  cent  sulphuric  acid  and  counter-stains 
with  Lbffler's  blue.  Here  the  granules  are  magenta,  the  protoplasm  blue. 
The  general  character  of  the  granules  thus  is  that  they  retain  the  first  stain 
more  intensely  than  the  rest  of  the  protoplasm  does. 

A  second  appearance  which  can  sometimes  be  seen  in  specimens  stained 
in  ordinary  ways  is  the  occurrence  of  a  concentration  of  the  protoplasm  at  each 
end  of  a  bacterium,  indicated  by  these  parts  being  deeply  stained.  These 
deeply  stained  parts  are  sometimes  called  polar  granules  (vide  Fig.  i,  No.  16, 
the  bacillus  most  to  the  left),  (German,  Polkb'rnchen  or  Polkorner). 

With  regard  to  the  significance  that  is  to  be  attached  to  such  appearances, 
much  depends  on  whether  they  are  constantly  present  under  all  circumstances, 
or  only  occasionally,  when  the  organism  is  grown  in  special  media  or  under 
special  growth  conditions.  Some  bacteria,  however  stained,  show  evidence 
of  having  the  protoplasm  somewhat  granular.  In  other  cases  this  granular 
condition  is  only  seen  when  the  organism  has  been  grown  under  bad  conditions, 
or  where  the  food  supply  is  becoming  exhausted.  Some  have  thought  that 
the  appearances  might  be  due  to  a  process  allied  to  mitosis,  and  might  signify 
approaching  division,  but  of  this  there  is  no  evidence. 

In  perfectly  healthy  and  young  bacteria,  moreover,  appearance  of  granule 
formation  and  of  vacuolation  may  be  accidentally  produced  by  physical  means 
in  the  occurrence  of  what  is  known  as  plasmolysis.  To  speak  generally,  when 
a  mass  of  protoplasm  surrounded  by  a  fairly  firm  envelope  of  a  colloidal  nature 


10  GENERAL   MORPHOLOGY   AND   BIOLOGY. 

is  placed  in  a  solution  containing  salts  in  greater  concentration  than  that  in 
which  it  has  previously  been  living,  then  by  a  process  of  osmosis  the  water 
held  in  the  protoplasm  passes  out  through  the  membrane,  and,  the  protoplasm 
retracting  from  the  latter,  the  appearance  of  vacuolation  is  presented.  Now  in 
making  a  dried  film  for  the  microscopic  examination  of  bacteria,  the  conditions 
necessary  for  the  occurrence  of  this  process  may  be  produced,  and  the  appear- 
ances of  vacuolation  and  of  Polkorner  may  thus  be  brought  about.  Plas- 
molysis  in  bacteria  has  been  extensively  investigated,1  and  has  been  found  to 
occur  in  some  species  more  readily  than  in  others.  We  may  conclude  that 
such  appearances  as  vacuolation  of  the  bacterial  protoplasm  and  Polkorner 
are  very  often  either  signs  of  degeneration,  like  the  metachromatic  granules, 
or  are  artificially  produced.  They  are  most  frequently  observed  in  old  or 
otherwise  enfeebled  cultures. 

Blitschli,  from  a  study  of  some  large  sulphur-containing  forms,  concludes 
that  the  greater  part  of  the  bacterial  cell  may  correspond  to  a  nucleus,  and 
that  this  is  surrounded  by  a  thin  layer  of  protoplasm,  which  in  the  smaller 
bacteria  escapes  notice,  unless  when,  as  in  the  bacilli,  it  can  be  made  out  at 
the  ends  of  the  cells.  Fischer,  it  may  be  said,  looks  on  the  appearances  seen 
in  Biitschli's  preparations  as  due  to  plasmolysis.  By  special  staining  methods, 
Nakanishi  believes  that  he  has  been  able  to  demonstrate,  undoubtedly,  nuclei 
in  a  number  of  bacterial  species.  His  work,  however,  has  not  yet  been 
confirmed. 

The  Chemical  Composition  of  Bacteria.  —  In  the  bodies  of 
bacteria  many  definite  substances  occur.  Some  bacteria  have 
been  described  as  containing  chlorophyll,  but  these  are  properly 
to  be  classed  with  the  schizophycese.  Sulphur  is  found  in  some 
of  the  higher  forms,  and  starch  granules  are  also  described  as 
occurring.  Many  species  of  bacteria,  when  growing  in  masses, 
are  brilliantly  coloured,  though  few  bacteria  associated  with  the 
production  of  disease  give  rise  to  pigments.  In  some  of  the 
organisms  classed  as  bacteria  a  pigment  named  bacterio-purpurin 
has  been  observed  in  the  protoplasm,  and  similar  intracellular 
pigments  probably  occur  in  some  of  the  larger  forms  of  the 
lower  bacteria  and  may  occur  in  the  smaller ;  but  it  is  usually 
impossible  to  determine  whether  the  pigment  occurs  inside  or 
outside  the  protoplasm.  In  many  cases,  for  the  free  produc- 
tion of  pigment  abundant  oxygen  supply  is  necessary ;  but 
sometimes,  as  in  the  case  of  spirillum  rubrum,  the  pigment  is 
best  formed  in  the  absence  of  oxygen.  Sometimes  the  faculty 
of  forming  it  may  be  lost  by  an  organism  for  a  time,  if  not 
permanently,  by  the  conditions  of  its  growth  being  altered. 

1  Consult  Fischer,  "  Untersuchungen  uber  Bakterien,"  Berlin,  1894;  "  Ueber 
den  Bau  der  Cyanophyceen  und  Bakterien,"  Jena,  1897. 


THE   CLASSIFICATION   OF   BACTERIA.  n 

Thus,  for  example,  if  the  B.  pyocyaneus  be  exposed  to  the 
temperature  of  42°  C.  for  a  certain  time,  it  loses  its  power  of 
producing  its  bluish  pigment.  Pigments  formed  by  bacteria 
often  diffuse  out  into,  and  colour,  the  medium  for  a  considerable 
distance  around. 

Comparatively  little  is  known  of  the  nature  of  bacterial  pigments.  Zopf, 
however,  has  found  that  many  of  them  belong  to  a  group  of  colouring  matters 
which  occur  widely  in  the  vegetable  and  animal  kingdoms,  viz.,  the  lipo- 
chromes.  These  lipochromes,  which  get  their  name  from  the  colouring 
matter  of  animal  fat,  include  the  colouring  matter  in  the  petals  of  Ranun- 
culaceae,  the  yellow  pigments  of  serum  and  of  the  yolks  of  eggs,  and  many 
bacterial  pigments.  The  lipochromes  are  characterised  by  their  solubility  in 
chloroform,  alcohol,  ether,  and  petroleum,  and  by  their  giving  indigo-blue 
crystals  with  strong  sulphuric  acid,  and  a  green  colour  with  iodine  dissolved 
in  potassium  iodide.  Though  crystalline  compounds  of  these  have  been 
obtained,  their  chemical  constitution  is  entirely  unknown,  and  even  their 
percentage  composition  is  disputed. 

Some  observations  have  been  made  on  the  chemical  structure 
of  bacterial  protoplasm.  Nencki  isolated  from  the  bodies  of 
certain  putrefactive  bacteria  proteid  bodies  which,  according  to 
Ruppel,  appear  to  have  been  allied  to  peptone,  and  which  cer- 
tainly differed  from  nucleo-proteids  in  not  containing  phos- 
phorus, but  many  of  the  proceids  isolated  by  other  chemists 
have  been  allied  in  their  nature  to  the  protoplasm  of  the  nuclei 
of  cells.  Buchner  in  certain  researches  obtained  bodies  of  this 
nature  allied  to  the  vegetable  caseins,  and  he  adduces  evidence 
to  show  that  it  is  to  these  that  the  characteristic  staining 
properties  are  due.  Various  observers  have  isolated  similar 
phosphorus-containing  proteids  from  different  bacteria.  Besides 
proteids,  however,  substances  of  a  different  nature  have  been 
isolated.  Thus  cellulose,  fatty  material,  chitin,  wax-like  bodies, 
and  other  substances  have  been  observed.  There  are  also 
found  various  mineral  salts,  especially  those  of  sodium,  potas- 
sium, and  magnesium.  The  amount  of  different  constituents 
varies  according  to  the  age  of  the  culture  and  the  medium  used 
for  growth,  and  certainly  great  variation  takes  place  in  the 
composition  of  different  species. 

The  Classification  of  Bacteria.  —  There  have  been  numerous 
schemes  set  forth  for  the  classification  of  bacteria,  the  funda- 
mental principle  running  through  all  of  which  has  been  the 
recognition  of  the  two  sub-groups  and  the  type  forms  mentioned 


12  GENERAL   MORPHOLOGY   AND    BIOLOGY. 

in  the  opening  paragraph  above.  In  the  attempts  to  still 
further  subdivide  the  group,  scarcely  two  systematists  are 
agreed  as  to  the  characters  on  which  sub-classes  are  to  be 
based.  Our  present  knowledge  of  the  essential  morphology 
and  relations  of  bacteria  is  as  yet  too  limited  for  a  really 
natural  classification  to  be  attempted.  To  prepare  for  the 
elaboration  of  the  latter,  Marshall  Ward  suggests  that  in  every 
species  there  should  be  studied  the  habitat,  best  food  supply, 
condition  as  to  gaseous  environment,  range  of  growth,  tem- 
perature, morphology,  and  life  history,  special  properties,  and 
pathogenicity. 

We  must  thus  be  content  with  a  provisional  and  incomplete 
classification.  We  have  said  that  the  division  into  lower  and 
higher  bacteria  is  recognised  by  all,  though,  as  in  every  other 
classification,  there  occur  transitional  forms.  In  subdividing 
the  bacteria  further,  the  forms  they  assume  constitute  at  present 
the  only  practicable  basis  of  classification.  The  lower  bacteria 
thus  naturally  fall  into  the  three  groups  mentioned,  the  cocci, 
bacilli,  and  spirilla,  though  the  higher  are  more  difficult  to  deal 
vwith.  Subsidiary,  though  important,  points  in  still  further  sub- 
division are  the  planes  in  which  fission  takes  place,  and  the 
presence  or  absence  of  spores.  The  recognition  of  actual 
species  is  often  a  matter  of  great  difficulty.  The  points  to  be 
observed  in  this  will  be  discussed  later  (p.  112). 

I.  The  Lower  Bacteria.1  —  These,  as  we  have  seen,  are 
minute  unicellular  masses  of  protoplasm  surrounded  by  an 
envelope,  the  total  vital  capacities  of  a  species  being  repre- 
sented in  every  cell.  They  present  three  distinct  type  forms, 
the  coccus,  the  bacillus,  and  the  spirillum ;  endogenous  sporula- 
tion  may  occur.  They  may  also  be  motile. 

I.  The  Cocci.  —  In  this  group,  the  cells  range  in  different 
species  from  .5  //,  to  2  M  in  diameter,  but  most  measure  about  I  /*. 
Before  division  they  may  increase  in  size  in  all  directions.  The 
species  are  usually  classified  according  to  the  method  of  division. 
If  the  cells  divide  only  in  one  axis,  and  through  the  consistency 
of  their  envelopes  remain  attached,  then  a  chain  of  cocci  will  be 
formed.  A  species  in  which  this  occurs  is  known  as  a  strepto- 
coccus. If  division  takes  place  irregularly  the  resultant  mass 

1  For  the  illustration  of  this  and  the  succeeding  systematic  paragraphs,  -vide 
Fig.  i. 


THE   CLASSIFICATION   OF   BACTERIA.  13 

may  be  compared  to  a  bunch  of  grapes,  and  the  species  is  often 
called  a  staphylococcus.  Division  may  take  place  in  two  axes  at 
right  angles  to  one  another,  in  which  case  cocci  adherent  to  each 
other  in  packets  of  four  (called  tetrads)  or  sixteen  may  be  found, 


156 


FIG.  i.  —  i.  Coccus.  2.  Streptococcus.  3.  Staphylococcus.  4.  Capsulated  diplococcus. 
5.  "  Biscuit  "-shaped  coccus.  6.  Tetrads.  7.  Sarcina  form.  8.  Types  of  bacilli  (1-8  are 
diagrammatic).  9.  Non-septate  spirillum  X  loco.  10.  Ordinary  spirillum  —  (a)  comma- 
shaped  element;  (b)  formation  of  spiral  by  comma-shaped  elements  X  loco.,  n.  Types 
of  spore  formation.  12.  Flagellated  bacteria.  13.  Changes  in  bacteria  produced  by  plas- 
molysis  (after  Fischer).  14.  Bacilli  with  terminal  protoplasm  (Butschli).  15.  (a)  Bacillus 
composed  of  five  protoplasmic  meshes;  (b)  protoplasmic  network  in  micrococcus  (Butschli). 
16.  Bacteria  containing  metachromatic  granules  (Ernst,  Neisser)  —  some  contain  polar 
granules.  17.  Beggiatoa  alba.  Both  filaments  contain  sulphur  granules  —  one  is  septate. 
18.  Thiothrix  tenuis  (Winogradski).  19.  Leptothrix  innominata  (Miller) .  20.  Cladothrix 
dichotoma  (Zopf ).  21.  Stfeptothrix  actinomyces  (Bostrom),  (a)  colony  under  low  power; 
(b~)  filament  showing  true  branching;  (c)  filament  containing  coccus-like  bodies;  (d)  fila- 
ment with  club  at  end. 


14  GENERAL   MORPHOLOGY   AND    BIOLOGY. 

the  former  number  being  the  more  frequent.  To  all  these  forms 
the  word  micrococcus  is  often  generally  applied.  The  individuals 
in  a  growth  of  micrococci  often  show  a  tendency  to  remain 
united  in  twos.  These  are  spoken  of  as  diplococci,  but  this  is 
not  a  distinctive  character,  since  every  coccus  as  a  result  of 
division  becomes  a  diplococcus,  though  in  some  species  the 
tendency  to  remain  in  pairs  is  well  marked.  The  adhesion  of 
cocci  to  one  another  depends  on  the  character  of  the  capsule. 
Often  this  has  a  well-marked  outer  limit  (micrococcus  tetragenus), 
sometimes  it  is  of  great  extent,  its  diameter  being  many  times 
that  of  the  coccus  (streptococcus  mesenterioides).  It  is  especially 
among  the  streptococci  and  staphylococci  that  the  phenomenon 
of  the  formation  of  arthrospores  is  said  to  occur.  In  none  of 
the  cocci  have  endogenous  spores  been  certainly  observed.  The 
number  of  species  of  the  streptococci  and  staphylococci  probably 
exceeds  150.  Usually  included  "in  this  group  are  coccus-like 
organisms  which  divide  in  three  axes  at  right  angles  to  one 
another.  These  are  usually  referred  to  as  sarcince.  If  the  cells 
are  lying  single  they  are  round,  but  usually  they  are  seen  in 
cubes  of  eight,  with  the  sides  which  are  in  contact  slightly 
flattened.  Large  numbers  of  such  cubes  may  be  lying  together. 
The  sarcinae  are,  as  a  rule,  rather  larger  than  the  other  members 
of  the  group.  Most  of  the  cocci  are  non-motile,  but  a  few  motile 
species  possessing  flagella  have  been  described. 

2.  Bacilli.  —  These  consist  of  long  or  short  cylindrical  cells, 
with  rounded  or  sharply  rectangular  ends,  usually  not  more  than 
I  /*  broad,  but  varying  very  greatly  in  length.  They  may  be 
motile  or  non-motile.  Where  flagella  occur,  these  maybe  dis- 
tributed all  round  the  organism,  or  only  at  one  or  both  of  the 
poles  {pseudomonas).  Several  species  are  provided  with  sharply 
marked  capsules  (B.  pneumonia).  In  many  species  endogenous 
sporulation  occurs.  The  spores  may  be  central  or  terminal, 
round,  oval,  or  spindle-shaped. 

Great  confusion  in  nomenclature  has  arisen  in  this  group  in  consequence 
of  the  different  artificial  meanings  assigned  to  the  essentially  synonymous 
terms  bacterium  and  bacillus.  Migula,  for  instance,  applies  the  former  term 
to  non-motile  species,  the  latter  to  the  motile.  Hueppe,  on  the  other  hand, 
calls  those  in  which  endogenous  sporulation  does  not  occur,  bacteria,  and 
those  where  it  does,  bacilli.  In  the  ordinary  terminology  Of  systematic  bac- 
teriology the  word  bacterium  has  been  almost  dropped,  and  is  reserved,  as  we 


THE   HIGHER   BACTERIA.  15 

have  done,  as  a  general  term  for  the  whole  group.  It  is  usual  to  call  all  the 
rod-shaped  varieties  bacilli.  And  until  the  botanists  themselves  agree  to 
adopt  a  common  standard  of  nomenclature,  it  is  preferable,  we  think,  to  retain 
the  use  of  the  older  system  throughout  this  work. 

3.  Spirilla. — These  consist  of  cylindrical  cells  more  or  less 
spiral  or  wavy.  Of  such  there  are  two  main  types.  In  one 
there  is  a  long  non-septate,  usually  slender,  wavy  or  spiral  thread 
(e.g.  spirillum  of  relapsing  fever,  Fig.  I,  No.  9).  In  the  other  type 
the  unit  is  a  short  curved  rod  (often  referred  to  as  of  a  "  comma  " 
shape).  When  two  or  more  of  the  latter  occur,  as  they  often  do, 
end  to  end  with  their  curves  alternating,  then  a  wavy  or  spiral 
thread  results.  An  example  of  this  is  the  cholera  microbe  (Fig. 
i,  No.  10).  This  latter  type  is  of  much  more  frequent  occurrence, 
and  contains  the  more  important  species.  Among  the  first  group 
motility  is  often  not  associated,  as  far  as  is  known,  with  the  pos- 
session of  flagella.  The  cells  here  apparently  move  by  an  undu- 
lating or  screw-like  contraction  of  the  protoplasm.  Most  of  the 
motile  spirilla,  however,  possess  flagella.  Of  the  latter  there 
may  be  one  or  two,  or  a  bunch  containing  as  many  as  twenty,  at 
one  or  both  poles.  Division  takes  place  as  among  the  bacilli,  and 
in  some  species  endogenous  sporulation  has  been  observed. 

Three  terms  are  used  in  dividing  this  group,  to  which  different  authors 
have  given  different  meanings.  These  terms  are  spirillum,  spirochaeta,  vibrio. 
Migula  makes  "  vibrio  "  synonymous  with  "  microspira,"  which  he  applies  to 
members  of  the  group  which  possess  only  one  or  two  polar  flagella ;  "  spiril- 
lum "  he  applies  to  similar  species  which  have  bunches  of  polar  flagella,  while 
"spirochaeta"  is  reserved  for  the  long  unflagellated  spiral  cells.  Hueppe 
applies  the  term  "spirochaeta"  to  forms  without  endospores,  "vibrio"  to 
those  with  endospores  in  which  during  sporulation  the  organism  changes  its 
form,  and  "  spirillum  *'  to  the  latter  when  no  change  of  form  takes  place  in 
sporulation.  Flugge,  another  systematist,  applies  "  spirochaeta  "  and  "  spiril- 
lum "  indiscriminately  to  any  wavy  or  corkscrew  form,  and  "  vibrio  "  to  forms 
where  the  undulations  are  not  so  well  marked.  It  is  thus  necessary,  in  de- 
nominating such  a  bacterium  by  a  specific  name,  to  give  the  authority  from 
whom  the  name  is  taken. 

II.  The  Higher  Bacteria.  —  These  show  advance  on  the  lower 
in  consisting  of  definite  filaments  branched  or  unbranched.  In 
most  cases  the  filaments  at  more  or  less  regular  intervals  are 
cut  by  septa  into  short  rod-shaped  or  curved  elements.  Such 
elements  are  more  or  less  interdependent  on  one  another,  and 
special  staining  methods  are  often  necessary  to  demonstrate  the 


16  GENERAL   MORPHOLOGY   AND    BIOLOGY. 

septa  which  demarcate  the  individuals  of  a  filament.  There  is 
further  often  a  definite  membrane  or  sheath  common  to  all  the 
elements  in  a  filament.  Not  only,  however,  is  there  this  close 
organic  relationship  between  the  elements  of  the  higher  bacteria, 
but  there  is  also  interdependence  of  function ;  for  example,  one 
end  of  a  filament  is  frequently  concerned  merely  in  attaching  the 
organism  to  some  other  object.  The  greatest  advance,  however, 
consists  in  the  setting  apart  among  most  of  the  higher  bacteria 
of  the  free  terminations  of  the  filaments  for  the  production  of 
new  individuals,  as  has  been  described  (p.  5).  There  are  vari- 
ous classes  under  which  the  species  of  the  higher  bacteria  are 
grouped ;  but  our  knowledge  of  them  is  still  somewhat  limited, 
as  many  of  the  members  have  not  yet  been  artificially  cultivated. 
The  beggiatoa  group  consists  of  free-swimming  forms,  motile  by 
undulating  contractions  of  their  protoplasm.  For  the  demon- 
stration of  the  rod-like  elements  of  the  filaments  special  staining 
is  necessary.  The  filaments  have  no  special  sheath,  and  the 
protoplasm  contains  sulphur  granules.  The  method  of  repro- 
duction is  doubtful.  The  thiothrix  group  resembles  the  last  in 
structure,  and  the  protoplasm  also  contains  sulphur  granules ; 
but  the  filaments  are  attached  at  one  end,  and  at  the  other  form 
gonidia.  The  leptothrix  group  resembles  closely  the  thiothrix 
group,  but  the  protoplasm  does  not  contain  sulphur  granules. 
In  the  cladothrix  group  there  is  the  appearance  of  branching, 
which,  however,  is  of  a  false  kind.  What  happens  is  that  a 
terminal  cell  divides,  and  on  dividing  again,  it  pushes  the  prod- 
uct of  its  first  division  to  one  side.  There  are  thus  two  termi- 
nal cells  lying  side  by  side,  and  as  each  goes  on  dividing,  the 
appearance  of  branching  is  given.  Here,  again,  there  is  goni- 
dium  formation ;  and  while  the  parent  organism  is  in  some  of 
its  elements  motile,  the  gonidia  move  by  means  of  flagella.  The 
highest  development  is  in  the  streptothrix  group,1  to  which  be- 
longs the  streptothrix  actinomyces,  or  the  actinomyces  bovis, 
an  important  pathogenic  agent.  Here  the  organism  consists  of 
a  felted  mass  of  non-septate  filaments,  in  which  true  dichotomous 
branching  occurs.  Under  certain  circumstances  threads  grow 
out  and  produce  chains  of  coccus-like  bodies  from  which  new 

1  Lachner-Sandoval  has  pointed  out  the  impropriety  of  the  employment  of  the 
term  "streptothrix,"  and  instead,  clearly  justifies  the  use  of  the  term  "actinomyces  " 
for  all  members  of  this  group. 


FOOD   SUPPLY.  ij 

individuals  can  be  reproduced.  Such  bodies  are  often  referred 
to  as  spores,  but  they  have  not  the  same  staining  reactions  nor 
resisting  powers  of  so  high  a  degree  as  ordinary  bacterial  spores. 
Sometimes  too  the  protoplasm  of  the  filaments  breaks  up  into 
bacillus-like  elements,  which  may  also  have  the  capacity  of 
originating  new  individuals.  In  the  streptothrix  actinomyces 
there  may  appear  a  club-shaped  swelling  of  the  membrane  at 
the  end  of  the  filament,  which  has  by  some  been  looked  on  as  an 
organ  of  fructification,  but  which  is  most  probably  a  product  of 
a  degenerative  change.  The  streptothrix  group,  though  its 
morphology  and  relationships  are  much  disputed,  may  be  looked 
on  as  a  link  between  the  bacteria  on  the  one  hand  and  the  lower 
fungi  on  the  other.  Like  the  latter,  the  streptothrix  forms  show 
the  felted  mass  of  non-septate  branching  filaments,  which  is 
usually  called  a  mycelium.  On  the  other  hand,  the  breaking  up 
of  the  protoplasm  of  the  streptothrix  into  coccus-  and  bacillus- 
like  forms,  links  it  to  the  other  bacteria. 

GENERAL  BIOLOGY  OF  THE  BACTERIA. 

There  are  five  prime  factors  which  must  be  considered  in  the 
growth  of  bacteria,  namely,  food  supply,  moisture,  relation  to 
gaseous  environment,  temperature,  and  light. 

Food  Supply. —  The  bacteria  are  chiefly  found  living  on  the 
complicated  organic  substances  which  form  the  bodies  of  dead 
plants  and  animals,  or  which  are  excreted  by  the  latter  while 
they  are  yet  alive.  Seeing  that,  as  a  general  rule,  many  bacteria 
grow  side  by  side,  the  food  supply  of  any  particular  variety  is, 
relatively  to  it,  altered  by  the  growth  of  the  other  varieties 
present.  It  is  thus  impossible  to  imitate  the  complexity  of  the 
natural  food  environment  of  any  species.  The  artificial  media 
used  in  bacteriological  work  may  therefore  be  poor  substitutes 
for  the  natural  supply.  In  certain  cases,  however,  the  conditions 
under  which  we  grow  cultures  may  be  better  than  the  natural 
conditions.  For  while  one  or  two  species  of  bacteria  growing 
side  by  side  may  favour  the  growth  of  the  other,  it  may  also 
in  certain  cases  hinder  it,  and,  therefore,  when  the  latter  is 
grown  alone  it  may  grow  better.  Most  bacteria  seem  to 
produce  excretions  which  are  unfavourable  to  their  own  vitality, 
for,  when  a  species  is  sown  on  a  mass  of  artificial  food  medium, 
it  does  not  in  the  great  majority  of  cases  go  on  growing  till  the 


18  GENERAL   MORPHOLOGY   AND   BIOLOGY. 

food  supply  is  exhausted,  but  soon  ceases  to  grow.  Effete 
products  diffuse  out  into  the  medium  and  prevent  growth. 
Such  diffusion  may  be  seen  when  the  organism  produces 
pigment,  e.g.  B.  pyocyaneus  growing  on  gelatin.  In  supplying 
artificial  food  for  bacterial  growth,  the  general  principle  ought 
to  be  to  imitate  as  nearly  as  possible  the  natural  surroundings, 
though  it  is  found  that  there  exists  a  considerable  adaptability 
among  organisms.  With  the  pathogenic  varieties  it  is  usually 
found  expedient  to  use  media  derived  from  the  fluids  of  the 
animal  body,  and  in  cases  where  bacteria  growing  on  plants  are 
being  studied,  infusions  of  the  plants  on  which  they  grow  are  fre- 
quently used.  Some  bacteria  can  exist  on  inorganic  food,  but 
most  require  organic  material  to  be  supplied.  Of  the  latter 
some  require  for  their  proper  nourishment  proteid  to  be  present, 
while  others  can  derive  their  nitrogen  from  such  a  non-proteid 
as  asparagin.  All  bacteria  require  nitrogen  to  be  present  in 
some  form,  and  many  require  to  derive  their  carbon  from 
carbohydrates.  Mineral  salts,  especially  sulphates,  chlorides, 
and  phosphates,  and  also  salts  of  iron,  are  necessary.  Occasion- 
ally special  substances  are  needed  to  support  life.  Thus  some 
species,  in  the  protoplasm  of  which  sulphur  granules  occur, 
require  sulphuretted  hydrogen  to  be  present.  In  nature  the 
latter  is  usually  provided  by  the  growth  of  other  bacteria.  When 
the  food  supply  of  a  bacterium  fails,  it  degenerates  and  dies. 
The  proof  of  death  lies  in  the  fact  that  when  it  is  transferred 
to  fresh  and  good  food  supply  it  does  not  multiply.  If  the 
bacterium  spores,  it  may  then  survive  the  want  of  food  for  a 
very  long  time.  It  may  here  be  stated  that  the  reaction  of  the 
food  medium  is  a  matter  of  great  importance.  Most  bacteria 
prefer  a  slightly  alkaline  medium,  and  some,  e.g.  the  cholera 
spirillum,  will  not  grow  in  the  presence  of  the  smallest  amount 
of  free  acid. 

Moisture.  —  The  presence  of  water  is  necessary  for  the  con- 
tinued growth  of  all  bacteria.  The  amount  of  drying  which 
bacteria  in  the  vegetative  stage  will  resist  varies  very  much  in 
different  species.  Thus  the  cholera  spirillum  is  killed  by  two  or 
three  hours'  drying,  while  the  staphylococcus  pyogenes  aureus 
will  survive  ten  days'  drying,  and  the  bacillus  diphtherias  still 
more.  In  the  case  of  spores  the  periods  are  much  longer. 
Anthrax  spores  will  survive  drying  for  several  years,  but  here 


TEMPERATURE.  ZQ 

again  moisture  enables  them  to  resist  longer  than  when  they  are 
quite  dry.  When  organisms  have  been  subjected  to  such  hostile 
influences,  even  though  they  survive  it  by  no  means  follows 
that  they  retain  all  their  vital  properties. 

Relation  to  Gaseous  Environment.  —  The  relation  of  bacteria 
to  the  oxygen  of  the  air  is  such  an  important  factor  in  the  life  of 
bacteria  that  it  enables  a  biological  division  to  be  made  among 
them.  Some  bacteria  will  only  live  and  grow  when  oxygen  is 
present.  To  these  the  title  of  obligatory  aerobes  is  given.  Other 
bacteria  will  only  grow  when  no  oxygen  is  present.  These  are 
called  obligatory  anaerobes.  To  still  other  bacteria  the  presence 
or  absence  of  oxygen  is  a  matter  of  indifference.  This  group 
might  theoretically  be  divided  into  those  which  are  preferably 
aerobes,  but  could  be  anaerobes,  and  those  which  are  preferably 
anaerobes,  but  could  be  aerobes.  As  a  matter  of  fact,  such 
differences  are  manifested  to  a  slight  degree,  but  all  such  organ- 
isms are  usually  grouped  as  facultative  anaerobes,  i.e.  prefer- 
ably aerobic  but  capable  of  existing  without  oxygen.  Examples 
of  obligatory  aerobes  are  B.  proteus  vulgaris,  B.  subtilis ;  of 
obligatory  anaerobes,  B.  tetani,  B.  oedematis  maligni,  while  the 
great  majority  of  pathogenic  bacteria  are  facultative  anaerobes. 
With  regard  to  anaerobes,  hydrogen  and  nitrogen  are  indifferent 
gases.  Many  anaerobes,  however,  do  not  flourish  well  in  an 
atmosphere  of  carbon  dioxide.  Very  few  experiments  have 
been  made  to  investigate  the  action  on  bacteria  of  gas  under 
pressure.  A  great  pressure  of  carbon  -dioxide  is  said  to  make 
the  B.  anthracis  lose  its  power  of  sporing,  but  it  seems  to  have 
no  effect  on  its  vitality  nor  on  that  of  the  B.  typhosus.  With 
the  bacillus  pyocyaneus,  it  is  said  to  destroy  life. 

Temperature.  —  For  every  species  of  bacterium  there  is  a 
temperature  at  which  it  grows  best.  This  is  called  the  "  opti- 
mum temperature."  There  is  also  in  each  case  a  maximum 
temperature  above  which  growth  does  not  take  place,  and  a 
minimum  temperature  below  which  growth  does  not  take  place. 
As  a  general  rule  the  optimum  temperature  is  about  the  temper- 
ature of  the  natural  habitat  of  the  organism.  For  organisms 
taking  part  in  the  ordinary  processes  of  putrefaction  the  temper- 
ature of  warm  summer  weather  (20°  to  24°  C.)  may  be  taken  as 
the  average  optimum,  while  for  organisms  normally  inhabiting 
animal  tissues  35°  to  39°  C.  is  a  fair  average.  The  lowest  limit  of 


20  GENERAL   MORPHOLOGY   AND    BIOLOGY. 

ordinary  growth  is  from  1 2°  to  14°  C,  and  the  upper  is  from  42°  to 
44°  C.  In  exceptional  cases  growth  may  take  place  as  low  as  5° 
C.  and  as  high  as  70°  C.  Some  organisms  which  grow  best  at  a 
temperature  of  from  60°  to  70°  C.  have  been  isolated  from  dung, 
the  intestinal  tract,  etc.  These  have  been  called  tkermophilic 
bacteria.  It  is  to  be  noted  that  while  growth  does  not  take  place 
below  or  above  a  certain  limit  it  by  no  means  follows  that  death 
takes  place  outside  such  limits.  Organisms  can  resist  cooling 
below  their  minimum  or  heating  beyond  their  maximum  without 
being  killed.  Their  vital  activity  is  merely  paralysed.  Espe- 
cially is  this  true  of  the  effect  of  cold  on  bacteria.  The  results 
of  different  observers  vary ;  but  if  we  take  as  an  example  the 
cholera  vibrio,  Koch  found  that  while  the  minimum  temperature 
of  growth  was  16°  C.,  a  culture  might  be  cooled  to  —32°  C.  with- 
out being  killed.  With  regard  to  the  upper  limit,  few  ordinary 
organisms  in  a  spore-free  condition  will  survive  a  temperature  of 
57°  C.,  if  long  enough  applied.  Many  organisms  lose  some  of 
their  properties  when  grown  at  unnatural  temperatures.  Thus 
many  pathogenic  organisms  lose  their  virulence  if  grown  above 
their  optimum  temperature,  and  some  chromogenic  forms,  most 
of  which  prefer  rather  low  temperatures,  lose  their  capacity  of 
producing  pigment,  e.g.  spirillum  rubrum. 

Effect  of  Light.  — Of  recent  years  much  attention  has  been 
paid  to  this  factor  in  the  life  of  bacteria.  Direct  sunlight  is 
found  to  have  a  very  inimical  effect.  One  observer  found 
that  an  exposure  of  dry  anthrax  spores  for  one  and  a  half  hours 
to  sunlight  killed  them.  When  they  were  moist,  a  much  longer 
exposure  was  necessary.  Typhoid  bacilli  were  killed  in  about 
one  and  a  half  hours,  and  similar  results  have  been  obtained 
with  many  other  organisms.  In  such  experiments  the  thickness 
of  the  medium  surrounding  the  growth  is  an  important  point. 
Death  takes  place  more  readily  if  the  medium  is  scanty  or  if  the 
organisms  are  suspended  in  water.  Any  fallacy  which  might 
arise  from  the  effect  of  the  heat  rays  of  the  sun  has  been  ex- 
cluded, though  light  plus  heat  is  more  fatal  than  light  alone. 
In  direct  sunlight  it  is  chiefly  the  green,  violet,  and,  it  may  be, 
the  ultra-violet  rays  which  are  fatal.  Diffuse  daylight  has  also 
a  bad  effect  upon  bacteria,  though  it  takes  a  much  longer  ex- 
posure to  do  serious  harm.  A  powerful  electric  light  is  as  fatal 
as  sunlight,  but  the  so-called  X-rays  are  quite  without  any 


CONDITIONS   AFFECTING    BACTERIA.  21 

germicidal  effect.  Here,  as  with  other  factors,  the  results  vary 
very  much  with  the  species  under  observation,  and  a  distinction 
must  be  drawn  between  a  mere  cessation  of  growth  and  the  con- 
dition of  actual  death.  Some  bacteria,  especially  occurring  on 
the  dead  bodies  of  fresh  fish,  are  phosphorescent. 

Conditions  affecting  the  Movements  of  Bacteria.  —  In  some 
cases  differences  are  observed  in  the  behaviour  of  motile  bacteria, 
contemporaneous  with  changes  in  their  life  history.  Thus,  in 
the  case  of  bacillus  subtilis,  movement  ceases  when  sporulation 
is  about  to  take  place.  On  the  other  hand,  in  the  bacillus  of 
symptomatic  anthrax,  movement  continues  while  sporulation  is 
progressing.  Under  ordinary  circumstances  motile  bacteria 
appear  not  to  be  constantly  moving  but  occasionally  to  rest. 
In  every  case  the  movements  become  more  active  if  the  tem- 
perature be  raised.  Most  interest,  however,  attaches  to  the 
fact  that  bacilli  may  be  attracted  to  certain  substances  and 
repelled  by  others.  Schenk,  for  instance,  observed  that  motile 
bacteria  were  attracted  to  a  warm  point  in  a  way  which  did  not 
occur  when  the  bacteria  were  dead  and  therefore  only  subject 
to  physical  conditions.  Most  important  observations  have  been 
made  on  the  attraction  and  repulsion  exercised  on  bacteria  by 
chemical  agents,  which  have  been  denominated  respectively 
positive  and  negative  chemiotaxis.  Pfeffer  investigated  this 
subject  in  many  lowly  organisms,  including  bacterium  termo 
and  spirillum  undula.  The  method  was  to  fill  with  the  agent 
a  fine  capillary  tube,  closed  at  one  end,  to  introduce  it  into  a 
drop  of  fluid  containing  the  bacteria  under  a  cover-glass,  and 
to  watch  the  effect  through  the  microscope.  The  general  result 
was  to  indicate  that  motile  bacteria  may  be  either  attracted  or 
repelled  by  the  fluid  in  the  tube.  The  effect  of  a  given  fluid 
differs  in  different  organisms,  and  a  fluid  chemiotactic  for  one 
organism  may  not  act  on  another.  Degree  of  concentration  is 
important,  but  the  nature  of  the  fluid  is  more  so.  Of  inorganic 
bodies  salts  of  potassium  are  the  most  powerfully  attracting 
bodies,  and  in  comparing  organic  bodies  the  important  factor 
is  the  molecular  constitution.  These  observations  have  been 
confirmed  by  Ali-Cohen,  who  found  that  while  the  vibrio  of 
cholera  and  the  typhoid  bacillus  were  scarcely  attracted  by 
chloride  of  potassium,  they  were  powerfully  influenced  by 
potato  juice.  Further,  the  filtered  products  or  the  growth  of 


22  GENERAL   MORPHOLOGY   AND   BIOLOGY. 

many  bacteria  have  been  found  to  have  powerful  chemiotactic 
properties.  It  is  evident  that  all  these  observations  have  a 
most  important  bearing  on  the  action  of  bacteria,  though  we  do 
not  yet  know  their  true  significance.  Corresponding  chemio- 
tactic phenomena  are  shown  also  by  certain  animal  cells,  e.g. 
leucocytes,  to  which  reference  is  made  below. 

The  Parts  played  by  Bacteria  in  Nature.  —  As  has  been  said, 
the  chief  effect  of  bacterial  action  in  nature  is  to  break  up  into 
more  simple  combinations  the  complex  molecules  of  the  organic 
substances  which  fojm  the  bodies  of  plants  and  animals,  or 
which  are  excreted  by  them,  In  some  cases  we  know  some  of 
the  stages  of  disintegration,  but  in  most  cases  we  know  only 
general  principles  and  sometimes  only  results.  In  the  case  of 
milk,  for  instance,  we  know  that  lactic  acid  is  produced  from 
the  lactose  by  the  action  of  the  bacillus  acidi  lactici  and  of 
other  bacteria,  and  that  from  urea  ammonium  carbonate  is 
produced  by  the  micrococcus  ureae.  That  the  very  compli- 
cated process  of  putrefaction  is  due  to  bacteria  is  absolutely 
proved,  for  any  organic  substance  can  be  preserved  indefinitely 
from  ordinary  putrefaction  by  the  adoption  of  some  method  of 
killing  all  bacteria  present  in  it,  as  will  be  afterwards  described. 
This  statement,  however,  does  not  exclude  the  fact  that  molecular 
changes  take  place  spontaneously  in  the  passing  of  the  organic 
body  from  life  to  death.  Many  processes  not  usually  referred  to 
as  putrefactive  are  also  bacterial  in  their  origin.  The  souring 
of  milk,  already  referred  to,  the  becoming  rancid  of  butter,  the 
ripening  of  cream  and  of  cheese,  are  all  due  to  bacteria. 

A  certain  comparatively  small  number  of  bacteria  have  been 
proved  to  be  the  causal  agents  in  some  disease  processes 
occurring  in  man,  animals,  and  plants.  This  means  that  the 
fluids  and  tissues  of  living  bodies  are,  under  certain  circum- 
stances, a  suitable  pabulum  for  the  bacteria  involved.  The 
effects  of  the  action  of  these  bacteria  are  analogous  to  those 
taking  place  in  the  action  of  the  same  or  other  bacteria  on  dead 
animal  or  vegetable  matter.  The  complex  organic  molecules 
are  broken  up  into  simpler  products.  We  shall  study  these 
processes  more  in  detail  later.  Meantime  we  may  note  that 
the  disease-producing  effects  of  bacteria  form  the  basis  of 
another  biological  division  of  the  group.  Some  bacteria  are 
harmless  to  animals  and  plants,  and  apparently  under  no 


THE   METHODS    OF   BACTERIAL   ACTION.  23 

circumstances  give  rise  to  disease  in  either.  These  are  known 
as  saprophytes.  They  are  normally  engaged  in  breaking  up 
dead  animal  and  vegetable  matter.  Others  normally  live  on 
or  in  the  bodies  of  plants  and  animals  and  produce  disease. 
These  are  known  as  parasitic  bacteria.  Sometimes  an  attempt 
is  made  to  draw  a  hard  and  fast  line  between  the  saprophytes 
and  the  parasites,  and  obligatory  saprophytes  or  parasites  are 
spoken  of.  This  is  an  erroneous  distinction.  Some  bacteria 
which  are  normally  saprophytes  can  produce  pathogenic  effects 
{e.g.  bacillus  cedematis  maligni),  and  it  is  consistent  with  our 
knowledge  that  the  best-known  parasites  may  have  been  derived 
from  saprophytes.  On  the  other  hand,  the  fact  that  most 
bacteria  associated  with  disease  processes,  and  proved  to  be 
the  cause  of  the  latter,  can  be  grown  in  artificial  media,  shows 
that  for  a  time  at  least  such  parasites  can  be  saprophytic.  As 
to  how  far  such  a  saprophytic  existence  of  disease-producing 
bacteria  occurs  in  nature,  we  are  in  many  instances  still 
ignorant. 

The  Methods  of  Bacterial  Action.  —  The  processes  which 
bodies  undergo  in  being  split  up  by  bacteria  depend,  first,  on 
the  chemical  nature  of  the  bodies  involved  and,  secondly,  on 
the  varieties  of  the  bacteria  which  are  acting.  The  destruction 
of  albuminous  bodies  which  is  mostly  involved  in  the  wide  and 
varied  process  of  putrefaction  can  be  undertaken  by  whole 
groups  of  different  varieties  of  bacteria.  The  action  of  the 
latter  on  such  substances  is  analogous  to  what  takes  place  when 
albumins  are  subjected  to  ordinary  gastric  and  intestinal  digestion. 
In  these  circumstances,  therefore,  the  production  of  albumoses, 
peptones,  etc.,  similar  to  those  of  ordinary  digestion,  can  be 
recognised  in  putrefying  solutions,  though  the  process  of  destruc- 
tion always  goes  further,  and  still  simpler  substances,  e.g.  indol, 
and,  it  may  be,  crystalline  bodies  of  an  alkaloidal  nature,  are 
the  ultimate  results.  The  process  is  an  exceedingly  complicated 
one  when  it  takes  place  in  nature,  and  different  bacteria  are 
probably  concerned  in  the  different  stages.  Many  other  bacteria, 
e.g.  some  pathogenic  forms,  though  not  concerned  in  ordinary 
putrefactive  processes,  have  a  similar  digestive  capacity.  When 
carbohydrates  are  being  split  up,  then  various  alcohols,  ethers, 
and  acids  are  produced.  During  bacterial  growth  there  is 
not  infrequently  the  abundant  production  of  such  gases  as 


24  GENERAL   MORPHOLOGY    AND   BIOLOGY. 

sulphuretted  hydrogen,  carbon  dioxide,  methane,  etc.  For  an 
exact  knowledge  of  the  destructive  capacities  of  any  particular 
bacterium  there  must  be  an  accurate  chemical  examination  of  its 
effects  when  it  has  been  grown  in  artificial  media  the  nature  of 
which  is  known.  The  precise  substances  it  is  capable  of  forming 
can  thus  be  found  out.  Many  substances,  however,  are  produced 
by  bacteria,  of  the  exact  nature  of  which  we  are  still  ignorant, 
for  example,  the  toxic  bodies  which  play  such  an  important  part 
in  the  action  of  many  pathogenic  species. 

Many  of  the  actions  of  bacteria  depend  on  the  production  by 
them  of  ferments  of  a  very  varied  nature  and  complicated  action. 
Thus  the  digestive  action  on  albumins  probably  depends  on  the 
production  of  a  peptic  ferment  analogous  to  that  produced  in 
the  animal  stomach.  Ferments  which  invert  sugar,  which  split 
sugars  up  into  alcohols  or  acids,  which  coagulate  casein,  which 
split  up  urea  into  ammonium  carbonate,  also  occur. 

Such  ferments  may  be  diffused  into  the  surrounding  fluid,  or 
be  retained  in  the  cells  where  they  are  formed.  Sometimes  the 
breaking  down  of  the  organic  matter  appears  to  take  place 
within,  or  in  the  immediate  proximity  of,  the  bacteria,  sometimes 
wherever  the  soluble  ferments  reach  the  organic  substances. 
And  in  certain  cases  the  ferments  diffused  out  into  the  sur- 
rounding medium  probably  break  down  constituents  of  the 
latter  to  some  extent,  and  prepare  them  for  a  further,  probably 
intracellular,  disintegration.  Thus  in  certain  putrefactions  of 
fibrin,  if  the  process  be  allowed  to  go  on  naturally,  the  fibrin 
dissolves  and  ultimately  great  gaseous  evolution  of  carbon 
dioxide  and  ammonia  takes  place,  but  if  the  bacteria,  shortly 
after  the  process  has  begun,  are  killed  or  paralysed  by  chloro- 
form, then  only  a  peptonisation  of  the  fibrin  occurs,  without 
the  further  splitting  up  and  gaseous  production  being  observed. 
That  a  purely  intracellular  digestion  may  take  place  is  illus- 
trated by  what  has  been  shown  to  occur  in  the  case  of  the  micro- 
coccus  ureae,  which  from  urea  forms  ammonium  carbonate  by 
adding  water  to  the  urea  molecule.  Here,  if  after  the  action  has 
commenced  the  bacteria  are  filtered  off,  no  further  production  of 
ammonium  carbonate  takes  place,  which  shows  that  no  ferment 
has  been  dissolved  out  into  the  urine.  If  now  the  bodies  of  the 
bacteria  be  extracted  with  absolute  alcohol  or  ether,  which  of 
course  destroys  their  vitality,  a  substance  is  obtained  of  the 


THE   METHODS   OF    BACTERIAL  ACTION.  25 

nature  of  a  ferment,  which,  when  added  to  sterile  urine,  rapidly 
causes  the  production  of  ammonium  carbonate.  This  ferment 
has  evidently  been  contained  within  the  bacterial  cells. 

In  considering  the  effects  of  bacteria  in  nature  it  must  be  recognised  that 
some  species  are  capable  of  building  up  -complex  substances  out  of  simple 
chemical  compounds.  Examples  of  these  are  found  in  the  bacteria  which 
in  the  soil  make  nitrogen  more  available  for  plant  nutrition  by  converting 
ammonia  into  nitrites  and  nitrates.  Winogradski,  by  using  media  containing 
non-nitrogenous  salts  of  magnesium,  potassium,  and  ammonium,  and  free  of 
organic  matter,  has  demonstrated  the  existence  of  forms  which  convert,  by 
oxidation,  ammonia  into  nitrites  and  of  other  forms  which  convert  these  nitrites 
into  nitrates.  Both  can  derive  their  necessary  carbon  from  alkaline  carbonates. 
Other  bacteria  or  organisms  allied  to  the  bacteria  exist  which  can  actually 
take  up  and  combine  into  new  compounds  the  free  nitrogen  of  the  air.  These 
are  found  in  the  tubercles  which  develop  on  the  rootlets  of  the  leguminosae. 
Without  such  organisms  the  tubercles  do  not  develop,  and  without  the  devel- 
opment of  the  tubercles  the  plants  are  poor  and  stunted.  Bacteria  thus  play 
an  important  part  in  the  enrichment  and  fertilisation  of  the  soil. 

The  Occurrence  of  Variability  among  Bacteria. — The  question  of  the  division 
of  the  group  of  bacteria  into  definite  species  has  given  rise  to  much  discussion 
among  vegetable  and  animal  morphologists,  and  at  one  time  very  divergent 
views  were  held.  Some  even  thought  that  the  same  species  might  at  one 
time  give  rise  to  one  disease,  —  at  another  time  to  another.  There  is,  how- 
ever, now  practical  unanimity  that  bacteria  show  as  distinct  species  as  the 
other  lower  plants  and  animals,  though,  of  course,  the  difficulty  of  defining 
the  concept  of  a  species  is  as  great  in  them  as  it  is  in  the  latter.  Still,  we  can 
say  that  among  the  bacteria  we  have  exhibited  (to  use  the  words  of  De  Bary) 
"  the  same  periodically  repeated  course  of  development  within  certain  empiri- 
cally determined  limits  of  variation  "  which  justifies,  among  higher  forms  of 
life,  a  species  to  be  recognised.  What  at  first  raised  doubts  as  to  the  occur- 
rence of  species  among  the  bacteria  was  the  observation  in  certain  cases  of 
what  is  known  as  pleomorphism.  By  this  is  meant  that  one  species  may 
assume  at  different  times  different  forms,  e.g.  appear  as  a  coccus,  a  bacillus, 
or  a  leptothrix.  Undoubtedly  many  of  the  cases  where  this  was  alleged  to 
have  been  observed  occurred  before  the  elaboration  of  the  modern  technique 
for  the  obtaining  of  pure  cultures,  but  at  the  present  day  there  are  cases  where 
evidence  appears  to  exist  of  the  occurrence  of  pleomorphism.  This  is  espe- 
cially the  case  with  certain  bacilli,  and  it  may  lead  to  such  forms  being  classed 
among  the  higher  bacteria.  Pleomorphism  is,  however,  a  rare  condition,  and 
with  regard  to  the  bacteria  as  a  whole  we  may  say  that  each  variety  tends  to 
conform  to  a  definite  type  of  structure  and  function  which  is  peculiar  to  it  and 
to  it  alone.  On  the  other  hand,  slight  variations  from  such  type  can  occur  in 
each.  The  size  may  vary  a  little  with  the  medium  in  which  the  organism  is 
growing,  and  under  certain  similar  *?onditions  the  adhesion  of  bacteria  to  each 
other  may  also  vary.  Thus  cocci,  which  are  ordinarily  seen  in  short  chains, 
may  grow  in  long  chains.  '  The  capacity  to  form  spores  may  be  altered,  and 


26  GENERAL   MORPHOLOGY   AND    BIOLOGY. 

such  properties  as  the  elaboration  of  certain  ferments  or  of  certain  pigments 
may  be  impaired.  Also  the  characters  of  the  growths  on  various  media  may 
undergo  variations.  As  has  been  remarked,  variation  as  observed  consists 
largely  in  a  tendency  in  a  bacterium  to  lose  properties  ordinarily  possessed, 
and  all  attempts  to  transform  one  bacterium  into  an  apparently  closely  allied 
variety  (such  as  the  B.  coli  into  the  B.  typhosus)  have  failed.  This  of  course 
does  not  preclude  the  possibility  of  one  species  having  been  originally  derived 
from  anothner  or  of  both  having  descended  from  a  common  ancestor,  but  we 
can  say  that  only  variations  of  an  unimportant  order  have  been  observed  to 
take  place,  and  here  it  must  be  remembered  that  in  many  cases  we  can  have 
forty-eight  or  more  generations  under  observation  within  twenty-four  hours. 


CHAPTER   II. 

METHODS   OF   CULTIVATION   OF   BACTERIA. 

Introductory.  —  In  order  to  study  the  characters  of  any  spe- 
cies of  bacterium  it  is  necessary  to  have  it  growing  apart  from 
every  other  species.  In  the  great  majority  of  cases  where  bac- 
teria occur  in  nature,  this  condition  is  not  fulfilled.  In  the  gen- 
eral processes  of  putrefaction  many  different  species  occur  all 
mingled  with  each  other.  Only  in  the  blood  and  tissues  in  some 
diseases  do  particular  species  occur  singly  and  alone.  We  usu- 
ally have,  therefore,  to  remove  a  bacterium  from  its  natural  sur- 
roundings and  grow  it  on  an  artificial  food  medium.  When  we 
have  succeeded  in  separating  it,  and  have  got  it  to  grow  on  a 
medium  which  suits  it,  we  are  said  to  have  obtained  a  pure  cul- 
ture. The  recognition  of  different  species  of  bacteria  depends, 
in  fact,  far  more  on  the  characters  presented  by  pure  cultures 
and  their  behaviour  in  different  food  media,  than  on  microscopic 
examination.  The  latter  in  most  cases  only  enables  us  to  refer 
a  given  bacterium  to  its  class.  Again,  in  inquiring  as  to  the 
possible  possession  of  pathogenic  properties  by  a  bacterium,  the 
obtaining  of  pure  cultures  is  absolutely  essential.  If  two  or 
more  different  organisms  be  present  together,  we  cannot  say 
that  any  one  of  them  is  the  cause  of  the  disease  in  question. 

To  obtain  pure  cultures,  then,  is  the  first  requisite  of  bacteri- 
ological research.  Now,  as  bacteria  are  practically  omnipresent, 
we  must  first  of  all  have  means  of  destroying  all  extraneous 
organisms  which  may  be  present  in  the  food  media  to  be  subse- 
quently used  for  growing  the  bacteria  we  wish  to  study,  in  the 
vessels  in  which  the  food  media  are  contained,  and  on  all  instru- 
ments which  are  to  come  in  contact  with  our  cultures.  The 
technique  of  this  destructive  process  is  called  sterilisation.  We 
must,  therefore,  study  the  methods  of  sterilisation.  The  growth 
of  bacteria  in  other  than  their  natural  surroundings  involves 
further  the  preparation  of  sterile  artificial  food  media,  and  when 
we  have  such  media  prepared  we  have  still  to  look  at  the  tech- 

27 


28  METHODS   OF   CULTIVATION    OF   BACTERIA. 

nique  of  the  separation  of  micro-organisms  from  mixtures  of 
these,  and  the  maintaining  of  pure  cultures  when  the  latter  have 
been  obtained.  We  shall  here  find  that  different  methods  are 
necessary  according  as  we  are  dealing  with  aerobes  or  anaerobes. 
Each  of  these  methods  will  be  considered  in  turn. 

THE  METHODS  OF  STERILISATION. 

To  exclude  extraneous  organisms,  all  food  materials,  glass 
vessels  containing  them,  wires  used  in  transferring  bacteria  from 
one  culture  medium  to  another,  instruments  used  in  making 
autopsies,  etc.,  must  be  sterilised.  These  objects  being  so 
different,  various  methods  are  necessary.  The  foods  comprise 
meat  infusions,  jellies,  potatoes,  etc.,  and  a  method  suitable  for 
their  sterilisation  evidently  may  not  be  suitable  for  the  sterilisa- 
tion of,  say,  a  glass  flask.  Bacteria  may  be  killed  by  various 
methods.  Many  chemicals  will  kill  them,  but  the  difficulty  of 
subsequently  removing  such  chemicals,  so  that  they  may  not 
interfere  with  the  growth  of  the  microbes  we  wish  to  cultivate, 
makes  their  use  inapplicable.  We  therefore  in  practice  take 
advantage  of  the  principle  that  all  bacteria  are  destroyed  by 
heat.  The  temperature  necessary  varies  with  different  bacteria, 
and  the  vehicle  of  heat  is  also  of  great  importance.  The  two 
vehicles  employed  are  hot  air  and  hot  water  or  steam.  The 
former  is  usually  referred  to  as  "dry  heat,"  the  latter  as  "moist 
heat."  As  showing  the  different  effects  of  the  two  vehicles, 
Koch  found,  for  instance,  that  the  spores  of  bacillus  anthracis, 
which  were  killed  by  moist  heat  at  100°  C,  in  one  hour, 
required  three  hours'  dry  heat  at  140°  C.  to  effect  death.  Both 
forms  of  heat  may  be  applied  at  different  temperatures ;  in 
the  case  of  moist  heat  above  100°  C.,  a  pressure  higher  than 
that  of  the  atmosphere  must  of  course  be  present. 

A.   Sterilisation  by  Dry  Heat. 

A.  (i)  Red  Heat  or  Dull  Red  Heat  — Red  heat  is  used  for 
the  sterilisation  of  the  platinum  needles,  which,  it  will  be  found, 
are  so  constantly  in  use.  A  dull  heat  is  used  for  cauteries,  the 
points  of  forceps,  and  may  be  used  for  the  incidental  sterilisa- 
tion of  small  glass  objects  (cover-slips,  slides,  occasionally  when 
necessary  even  test-tubes),  care  of  course  being  taken  not  to  melt 
the  glass.  The  heat  is  obtained  by  an  ordinary  Bunsen  burner. 


STERILISATION    BY    MOIST    HEAT. 


FIG.  2. — -Hot-air  steriliser. 


A.  (2)  Sterilisation  by  Dry  Heat  in  a  Hot-air  Chamber. - 

The  chamber  (Fig.  2)  consists  of  an  outer  and  inner  case  of 
sheet  iron.  In  the  bottom  of  the  outer  there  is  a  large  hole. 
A  Bunsen  is  lit  beneath  this,  and 
thus  plays  on  the  bottom  of  the 
inner  case,  round  all  of  the  sides  of 
which  the  hot  air  rises  and  escapes 
through  holes  in  the  top  of  the 
outer  case.  A  thermometer  passes 
down  into  the  interior  of  the  cham- 
ber, halfway  up  which  its  bulb 
should  be  situated.  It  is  found,  as 
a  matter  of  experience,  that  an  ex- 
posure in  such  a  chamber  for  one 
hour  to  a  temperature  of  170°  C., 
is  sufficient  to  kill  all  the  organisms 
which  usually  pollute  articles  in  a 
bacteriological  laboratory,  though 
circumstances  might  arise  where  this  would  be  insufficient. 
This  means  of  sterilisation  is  used  for  the  glass  flasks,  test-tubes, 
plates,  Petri's  dishes,  the  use  of  which  will  be  described.  Such 
pieces  of  apparatus  are  thus  obtained  sterile  and  dry.  It  is 
advisable  to  put  glass  vessels  into  the  chamber  before  heating  it, 
and  to  allow  them  to  stand  in  it  after  sterilisation  till  the  tem- 
perature falls.  Sudden  heating  or  cooling  is  apt  to  cause  glass 
to  crack.  The  method  is  manifestly  unsuitable  for  food  media. 

B.   Sterilisation  by  Moist  Heat. 

B.  (i)  By  Boiling. — The  boiling  of  a  liquid  for  five  minutes 
is  sufficient  to  kill  ordinary  germs  if  no  spores  be  present,  and 
this  method  is  useful  for  sterilising  distilled  or  tap  water  which 
may  be  required  in  various  manipulations.     It  is  best  to  sterilise 
knives  and  instruments  used  in  autopsies  by  boiling  in  water  to 
which  a  little  sodium  carbonate  has  been  added  to  prevent  rust- 
ing.    Twenty  minutes'  boiling  will  here  be  sufficient.     The  boil- 
ing of  any  fluid  at  100°  C.  for  one  and  a  half  hours  will  ensure 
sterilisation  under  almost  any  circumstances. 

B.  (2)  By  Steam  at  100°  C. —  This  is  by  far  the  most  useful 
means  of  sterilisation.  It  may  be  accomplished  in  an  ordinary 
potato  steamer  placed  on  a  kitchen  pot.  The  apparatus  ordi- 


METHODS    OF   CULTIVATION   OF   BACTERIA. 


FIG.  3.  —  Koch's 
steam  steriliser. 


narily  used  is  "  Koch's  steam  steriliser"  (Fig.  3).     This  consists 
of  a  tall  metal  cylinder  on  legs,  provided  with  a  lid,  and  covered 
c  externally   by   some   bad   conductor   of    heat, 

such   as  felt   or   asbestos.     A   perforated   tin 
diaphragm  is  fitted  in  the  interior  at  a  little 
distance  above  the  bottom,  and  there  is  a  tap 
at  the  bottom  by  which  water  may   be   sup- 
plied or  withdrawn.     If  water  to  the  depth  of 
3  inches  be  placed  in  the  interior  and  heat 
applied,  it  will   quickly  boil,   and   the  steam 
streaming  up  will  surround  any  flask  or  other 
object  standing  on  the  diaphragm.     Here  no 
evaporation  takes  place  from  any  medium  as 
it    is    surrounded    during    sterilisation    by    an 
atmosphere  saturated  with  water  vapour.     It 
is  convenient  to  have  the  cylinder  tall  enough 
to  hold  a  litre  flask  with  a  funnel  7  inches  in 
diameter   standing   in   its   neck.     The  funnel 
may  be  supported  by  passing  its  tube  through  a  second  per- 
forated   diaphragm   placed   in    the   upper   part   of    the   steam 
chamber.     With  such  a  "  Koch  "  in  the  laboratory  a  hot-water 
filter  is  not  needed.     An  even  more  serviceable  steriliser  is  that 
known  as  the  Arnold  steam  steri- 
liser, which,  by  its  peculiar  con- 
struction, effects  a  greater  saving 
in  the  time  necessary  to  develop 
steam  than  does  a  similar  sized 
Koch   apparatus.     As  has   been 
said,  one  and  a  half  hours'  steam- 
ing will  sterilise  any  medium,  but 
in  the  case  of  media  containing 
gelatin  such  an  exposure  is  not 
practicable,  as,  with  long  boiling, 
gelatin  tends  to  lose  its  physical 
property    of    solidification.     The 
method  adopted  in  this  case  is  to 
steam  for  a  quarter  of  an  Jiour 
on  each  of  three  succeeding  days. 

This  is  a  modification  of  what  is  known  as  "  Tyndall's  intermit- 
tent sterilisation."     The  fundamental  principle  of  this  method  is 


FIG.  4.  —  The  Arnold  steam-steriliser. 


STERILISATION   BY   MOIST   HEAT. 


that  all  bacteria  in  a  non-spored  form  are  killed  by  the  tempera- 
ture of  boiling  water,  while  if  in  a  spored  form  they  may  not 
be  thus  killed.  Thus  by  the  sterilisation  on  the  first  day  all  the 
non-spored  forms  are  destroyed  —  the  spores  remaining  alive. 
During  the  twenty-four  hours  which  intervene  before  the  next 
heating,  these  spores,  being  in  a  favourable  medium,  are  likely 
to  assume  the  non-spored  form.  The  next  heating  kills  these. 
In  case  any  may  still  not  have  changed  their  spored  form,  the 
process  is  repeated  on  a  third  day.  Experience  shows  that 
usually  the  medium  can  now  be  kept  indefinitely  in  a  sterile  con- 
dition. Steam  at  100°  C.  is  therefore  under  most  conditions 
available  for  the  sterilisation  of  all  ordinary  media.  In  using 
the  Koch's  steriliser,  especially  when  a  large  bulk  of  medium  is 
to  be  sterilised,  it  is  best  to  put  the  media  in  while  the  appa- 
ratus is  cold,  in  order  to  make  certain  that  the  whole  of  the  food 
mass  reaches  the  temperature  of  100°  C.,  and  it  is  preferable 
to  prolong  the  period  of  exposure  to  half  an  hour,  always  reck- 
oning from  the  time  boiling  commences  in  the  water  in  the 
steriliser. 

If  we  wish  to  use  such  a  substance  as  blood  serum  as  a  me- 
dium, the  albumin  would  be  coagulated  by  a 
temperature  of  100°  C.     Therefore  other  means 
have  to  be  adopted  in  this  case. 

B.  (3)  Sterilisation  by  Steam  at  High  Pres- 
sure. —  This  is  the  most  rapid  and  effective 
means  of  sterilisation.  It  is  effected  in  an 
autoclave  (Fig.  5).  This  is  a  gun-metal  cylin- 
der on  legs,  the  top  of  which  is  fastened  down 
with  screws  and  nuts  and  is  furnished  with  a 
safety  valve,  pressure-gauge,  and  a  hole  for 
thermometer.  As  in  the  Koch's  steriliser,  the 
contents  are  supported  on  a  perforated  dia- 
phragm. The  source  of  heat  is  a  large  Bunsen 
beneath.  The  temperature  employed  is  usually 
115°  C.  or  120°  C.  To  boil  at  115°  C.,  water  re- 
quires a  pressure  of  about  23  Ib.  to  the  square 
inch  (i.e.  8  Ib.  plus  the  1 5  Ib.  of  ordinary  atmos- 
pheric pressure).  To  boil  at  I2O°C,  a  pressure 
of  about  30  Ib.  (i.e.  15  Ib.  plus  the  usual  pressure)  is  necessary. 
In  such  an  apparatus  the  desired  temperature  is  maintained  by 

OF  THE 
UNIVERSITY 

OF 


00     0000 


FIG.  5.  —  Autoclave. 

a,  safety  valve. 

b,  blow-off  pipe. 

c ,  gauge. 


METHODS   OF   CULTIVATION    OF   BACTERIA. 


adjusting  the  safety  valve  so  as  to  blow  off  at  the  correspond- 
ing pressure.  One  exposure  of  media  (when  in  small  bulk)  to 
the  latter  temperature  for  seven  minutes  is  sufficient  to  kill  all 
organisms  or  spores,  but  if  the  bulk  is  great,  then  it  is  advisable 
to  prolong  the  exposure  to  fifteen  minutes.  Here,  again,  care 
must  be  taken  when  gelatin  is  to  be  sterilised.  It  must  not  be 
exposed  to  a  temperature  above  105°  C.,  and  must  be  sterilised 
by  the  intermittent  method.1  Certain  precautions  are  necessary 
in  using  the  autoclave.  In  all  cases  it  is  necessary  to  allow 
the  apparatus  to  cool  well  below  100°  C.  before  opening  it  or 
allowing  steam  to  blow  off,  otherwise  there  will  be  a  sudden  de- 
velopment of  steam  when  the  pressure  is  removed,  and  fluid 
media  will  be  blown  out  of  the  flasks.  Sometimes  the  instru- 
ment is  not  fitted  with  a  thermometer.  In  this  case  care  must 
be  taken  to  expel  all  the  air  initially  present,  otherwise  a  mix- 
ture of  air  and  steam  being  present,  the  pressure  read  off  the 
gauge  cannot  be  accepted  as  an  indication  of  the  temperature. 
Further,  care  must  be  taken  to  ensure 
the  presence  of  a  residuum  of  water 
when  steam  is  fully  up,  otherwise  the 
steam  is  superheated,  and  the  pressure 
on  the  gauge  again  does  not  indicate 
the  temperature  correctly. 

B.  (4)  Sterilisation  at  Low  Tempera- 
tures. —  Most  organisms  in  a  non-spored 
form  are  killed  by  a  prolonged  exposure 
to  a  temperature  of  57°  C.     This  fact  has 
been  taken  advantage  of  for  the  sterili- 
sation of  blood  serum,  which  will  coagu- 
late if  exposed  to  a  temperature  above 
that  point.     Such  a  medium  is  sterilised 
on  Tyndall's  principle  by  exposing  it  for 
an  hour  at   57°  C.  for  eight  consecutive 
days,  it  being  allowed  to  cool  in  the  in- 
terval to  the  room  temperature.     The  apparatus  shown  in  Fig.  6 
is  a  small  hot-water  jacket  heated  by  a  Bunsen  placed  beneath 
it,  the   temperature   being  controlled  by  a  gas  regulator.     To 

1  This  medium  will  retain  its  gelatinising  power  perfectly  well  when  sterilised  for 
five  minutes  in  the  autoclave,  if,  on  removal  therefrom,  it  be  at  once  placed  in  the  ice- 
chest  to  cool. 


FlG.  6. —  Steriliser  lor  blood 
serum. 


PREPARATION  OF  CULTURE  MEDIA.         33 

ensure  that  the  temperature  all  around  shall  be  the  same,  the  lid 
also  is  hollow  and  filled  with  water,  and  there  is  a  special  gas 
burner  at  the  side  to  heat  it.  This  is  the  form  originally  used, 
but  serum  sterilisers  are  now  constructed  in  which  the  test-tubes 
are  placed  in  the  sloped  position,  and  in  which  inspissation  (vide 
p.  43)  can  afterwards  be  performed  at  a  higher  temperature. 

THE  PREPARATION  OF  CULTURE  MEDIA. l 

The  general  principle  to  be  observed  in  the  artificial  culture 
of  bacteria  is  that  the  medium  used  should  approximate  as 
closely  as  possible  to  that  on  which  the  bacterium  grows  natu- 
rally. In  the  case  of  pathogenic  bacteria  the  medium  therefore 
should  resemble  the  juices  of  the  body.  The  serum  of  the  blood 
satisfies  this  condition  and  is  often  used,  but  its  application  is 
limited  by  the  difficulties  in  its  preparation  and  preservation. 
Other  media  have  been  found  which  can  support  the  life  of  all 
the  pathogenic  bacteria  isolated.  These  consist  of  proteids  or 
carbohydrates  in  a  fluid,  semi-solid,  or  solid  form,  in  a  transpar- 
ent or  opaque  condition.  The  advantage  of  having  a  variety  of 
media  lies  in  the  fact  that  growth  characters  on  particular  media, 
non-growth  on  some  and  growth  on  others,  etc.,  constitute  spe- 
cific differences  which  are  valuable  in  the  identification  of  bac- 
teria. The  most  commonly  used  media  have  as  their  basis  a 
watery  extract  of  meat.  Most  bacteria  in  growing  in  such  an 
extract  cause  only  a  grey  turbidity.  A  great  advance  resulted 
when  Koch,  by  adding  to  it  gelatin,  provided  a  transparent  solid 
medium  in  which  growth  characteristics  of  particular  bacteria 
become  evident.  Many  organisms,  however,  grow  best  at  a 
temperature  at  which  this  nutrient  gelatin  melts,  and  therefore 
another  gelatinous  substance  of  vegetable  origin,  called  agar, 
which  does  not  melt  below  98°  C,  was  substituted.  Bouillon 
made  from  meat  extract,  gelatin,  and  agar  media,  and  the  modi- 
fications of  these,  constitute  the  chief  materials  in  which  bacteria 
are  grown. 

1  The  student  is  strongly  advised  to  make  himself  familiar  with  the  more  elaborate 
technique  laid  down  by  the  Bacteriological  Committee  of  the  American  Public  Health 
Association,  which  has  been  adopted  in  America  as  the  standard  of  procedure  in 
bacteriological  investigation.  The  technique  as  outlined  in  the  following  pages  will 
be  found,  however,  to  be  more  suitable  to  the  necessities  of  the  work  of  large  classes, 
or  where  facilities  for  the  prosecution  of  advanced  work  are  lacking. 


34  METHODS    OF   CULTIVATION   OF   BACTERIA. 

Preparation  of  Meat  Infusion. 

The  flesh  of  the  ox,  calf,  or  horse  is  usually  employed. 
Horse-flesh  has  the  advantage  of  being  cheaper  and  containing 
less  fat  than  the  others  ;  though  generally  quite  suitable,  it  has 
the  disadvantage  for  certain  purposes  of  containing  a  larger 
proportion  of  fermentable  sugar.  The  flesh  must  be  freed 
from  fat,  and  finely  minced.  To  a  pound  of  mince  add  1000 
c.c.  distilled  water,  and  mix  thoroughly  in  a  shallow  dish.  Set 
aside  in  a  cool  place  for  twenty-four  hours.  Skim  off  any  fat 
present,  removing  the  last  traces  by  stroking  the  surface  of  the 
fluid  with  pieces  of  filter  paper.  Place  a  clean  linen  cloth  over 
the  mouth  of  a  larger  filter  funnel,  and  strain  the  fluid  through 
it  into  a  flask.  Pour  the  minced  meat  into 
the  cloth,  and  gathering  up  the  edges  of  the 
latter  in  the  left  hand,  squeeze  out  the  juice 
still  held  back  in  the  contained  meat.  Finish 
this  expression  by  putting  the  cloth  and  its 
contents  into  a  meat  press  (Fig.  7),  similar 
to  that  used  by  pharmacists  in  preparing  ex- 
tracts ;  thus  squeeze  out  the  last  drops.  The 
resulting  sanguineous  fluid  contains  the  solu- 
ble albumins  of  the  meat,  the  soluble  salts, 
extracts,  extractives,  and  colouring  matter, 
chiefly  haemoglobin.  It  is  now  boiled  thoroughly  for  two  hours, 
by  which  process  the  albumins  coagulable  by  heat  are  coagulated. 
Strain  now  through  a  clean  cloth,  boil  for  another  half-hour, 
and  filter  through  white  Swedish  filter  paper  (best,  C.  Schleicher 
u.  Schull,  No.  595).  Make  up  to  1000  c.c.  with  distilled  water. 
The  resulting  fluid  ought  to  be  quite  transparent,  of  a  yellowish 
colour  without  any  red  tint.  If  there  is  any  redness,  the  fluid 
must  be  reboiled  and  filtered  till  this  colour  disappears,  other- 
wise in  the  later  stages  it  will  become  opalescent.  A  large 
quantity  of  the  infusion  may  be  made  at  a  time,  and  what  is  not 
immediately  required  is  put  into  a  large  flask,  the  neck  plugged 
with  cotton  wool,  and  the  whole  sterilised  by  methods  B  (2)  or 
(3).  This  infusion  contains  very  little  albuminous  matter,  and 
consists  chiefly  of  the  soluble  -salts  of  the  muscle,  certain  ex- 
tractives, and  altered  colouring  matters,  along  with  any  slight 
traces  of  soluble  proteid  not  coagulated  by  heat.  It  is  of  acid 


PEPTONE    BOUILLON   MEDIA.  35 

reaction.  We  have  now  to  see  how,  by  the  addition  of  proteid 
and  other  matter,  it  may  be  transformed  into  proper  culture 
media.  Another  and  equally  reliable  method  of  making  meat 
infusion,  in  which  a  great  saving  of  time  is  accomplished,  is  by 
dissolving  2\  grammes  of  Liebig's  extract  of  meat  in  1000  c.c. 
of  boiling  water. 

i.  Bouillon  Media.  —  These  consist  of  meat  infusion  with 
the  addition  of  certain  substances  to  render  them  suitable  for 
the  growth  of  bacteria.  • 

i  (a).  Peptone  Broth  or  Bouillon. — This  has  the  composi- 
tion :  — 

Meat  infusion       ....     1000  c.c. 
Sodium  chloride  ...  5  grms. 

Peptone  albumin  .         .         .         10     „ 

Boil  till  the  ingredients  are  quite  dissolved,  and  neutralise  with 
a  4  per  cent  solution  of  sodium  hydrate.  This  is  done  by  add- 
ing cautiously  a  cubic  centimetre  or  two  of  the  sodium  hydrate 
solution,  stirring  well  the  while,  and  testing  the  reaction  by 
means  of  phenol-phthaleine  paper,  proceeding  until  the  latter 
strikes  a  well-defined  rose-pink  colour,  thus  indicating  the  point 
of  beginning  alkalinity.  Should,  however,  the  color  be  deeper, 
approaching  a  madder,  then  the  alkalinity  can  be  decreased  by 
adding  enough  of  a  5  per  cent  hydrochloric  acid  solution  until 
the  desired  colour  tint  is  acquired.  To  prevent  the  subsequent 
precipitation  of  phosphates  and  other  matters  in  the  broth  after 
autoclaving  in  tubes,  it  is  recommended  to  autoclave  the  whole 
of  the  broth  after  adjusting  the  reaction,  allowing  it  to  become 
cold,  and  then  filter  it,  when  it  may  be  tubed  and  again  auto- 
claved  with  rro  fear  of  any  subsequent  clouding  of  the  medium. 
This  method  of  neutralisation  is  to  be  recommended  for  all 
ordinary  work. 

In  this  medium  the  place  of  the  original  albumins  of  the  meat  is  taken  by 
peptone,  a  soluble  proteid  not  coagulated  by  heat.  Here  it  may  be  remarked 
that  the  commercial  peptone  albumin  is  not  pure  peptone,  but  a  mixture  of 
albumoses  (see  footnote,  p.  172)  with  a  variable  amount  of  pure  peptone.  The 
addition  of  the  sodium  chloride  is  necessitated  by  the  fact  that  alkalinisation 
precipitates  some  of  the  phosphates  and  carbonates  present.  Experience  has 
shown  that  sodium  chloride  can  quite  well  be  substituted.  The  reason  for  the 
alkalinisation  is  that  it  is  found  that  most  bacteria  grow  best  on  a  medium 
slightly  alkaline  to  litmus.  Some,  e.g.  the  cholera  vibrio,  will  not  grow  at  all 
on  even  a  slightly  acid  medium. 


36  METHODS    OF    CULTIVATION    OF   BACTERIA. 

Standardisation  of  Reaction  of  Media.  —  While  the  above  pro- 
cedure of  dealing  with  the  reaction  of  a  medium  is  sufficient 
for  ordinary  work,  it  has  been  thought  advisable  to  have  a 
more  exact  method  for  making  media  to  be  used  in  growing 
organisms,  the  growth  characteristics  of  which  are  to  be  de- 
scribed for  systematic  purposes.  Such  a  method  should  also 
be  used  in  studying  the  changes  in  reaction  produced  in  a  me- 
dium by  the  growth  of  bacteria.  It,  however,  involves  consid- 
erable difficulty,  and  should  not  be  undertaken  by  the  beginner. 
It  entails  the  preparation  of  solutions  of  acid  and  alkali  which 
may  be  used  for  determining  the  original  reaction  of  the  me- 
dium, and  for  accurately  making  it  of  a  definite  degree  of  alkalin- 
ity. Normal1  and  decinormal  solutions  of  sodium  hydrate  and 
hydrochloric  acid  are  used. 

Preparation  of  Standard  Solutions.  —  The  first  requisites  here  are  normal 
solutions  of  acid  and  alkali.  The  latter  is  prepared  as  follows  :  85  grammes 
of  pure  sodium  bicarbonate  are  heated  to  dull  redness  for  ten  minutes  in  a 
platinum  vessel  and  allowed  to  cool  in  an  exsiccator;  just  over  54  grammes 
of  sodium  carbonate  should  now  be  present.  Any  excess  is  quickly  removed, 
and  the  rest  being  dissolved  in  one  litre  of  distilled  water,  a  normal  solution 
is  obtained.  A  measured  quantity  is  placed,  in  a  porcelain  dish,  and  a  few 
drops  of  a  .5  per  cent  solution  of  phenol-phthaleine  in  50  per  cent  alcohol  is 
added  to  act  as  indicator.  The  alkali  produces  in  the  latter  a  brilliant  rose 
pink,  which,  however,  disappears  on  the  least  excess  of  acid  being  present. 
The  mixture  is  boiled  and  a  solution  of  hydrochloric  acid  of  unknown  strength 
is  run  into  the  dish  from  a  burette  till  the  colour  goes,  and  does  not  return 
after  very  thorough  stirring.  The  strength  of  the  acid  can  then  be  calculated, 
and  a  normal  solution  can  be  obtained.  From  these  two  solutions  any 
strength  of  acid  or  alkali  (such  as  the  decinormal  solution  of  NaOH  mentioned 
below)  may  be  derived. 

As  Eyre  has  suggested,  the  reaction  of  a  medium  may  be 
conveniently  expressed  by  the  sign  +  or  —  to  indicate  acid  or 
alkaline  respectively,  and  a  number  to  indicate  the  number  of 
cubic  centimetres  of  normal  acid  or  alkaline  solution  necessary 

1 A  "  normal "  solution  of  any  salt  is  prepared  by  dissolving  an  "  equivalent " 
weight  in  grammes  of  that  salt  in  a  litre  of  distilled  water.  If  the  metal  of  the  salt 
be  monovalent,  i.e.  if  it  be  replaceable  in  a  compound  by  one  atom  of  hydrogen  (e.g. 
sodium),  an  equivalent  is  the  molecular  weight  in  grammes.  In  the  case  of  NaCl,  it 
would  be  58.5  grammes  (atomic  weight  of  Na  =  23,  of  €1  =  35.5).  ^  tne  metal  be 
bivalent,  i.e.  requiring  two  atoms  of  H  for  its  replacement  in  a  compound  (e.g.  cal- 
cium), an  equivalent  is  the  molecular  weight  in  grammes  divided  by  two.  Thus  in 
the  case  of  CaCl2  an  equivalent  would  be  55.5  grammes  (atomic  weight  of  Ca  =  4O, 
of  Cl2  =  i). 


STANDARDISING   REACTION   OF   MEDIA.  37 

to  make  a  litre  of  the  medium  neutral  to  phenol-phthaleine. 
Thus,  for  example,  "  reaction  =  —15,"  will  mean  that  the  me- 
dium is  alkaline,  and  requires  15  c.c.  of  normal  HC1  to  make  a 
litre  neutral.  It  has  been  found  that  when  a  medium  such  as 
bouillon  reacts  neutral  to  litmus,  its  reaction  to  phenol-phtha- 
leine, according  to  the  above  standard,  is  on  the  average  +  25. 
Now  as  litmus  was  originally  introduced  by  Koch,  and  as 
nearly  all  bacterial  research  has  been  done  with  media  tested  by 
litmus,  it  is  evidently  difficult  to  say  exactly  what  precise  degree 
of  alkalinity  is  the  optimum  for  bacterial  growth.  It  is  probably 
safe  to  say,  however,  that  when  a  medium  has  been  rendered 
neutral  to  phenol-phthaleine  by  the  addition  of  NaOH,  the 
optimum  degree  is  attained  by  the  addition  of  from  10  to  15  c.c. 
of  normal  HC1  per  litre,  i.e.  the  optimum  reaction  is  -f  10  to  +15. 
In  other  words,  the  optimum  reaction  for  bacterial  growth  lies, 
as  Fuller  has  pointed  out,  about  midway  between  the  neutral 
point  indicated  by  phenol-phthaleine  and  the  neutral  point  in- 
dicated by  litmus.1 

The  only  objection  to  the  use  of  phenol-phthaleine  is  that  its 
action  is  somewhat  vitiated  if  free  CO2  be  present.  This  can 
be  completely  obviated  as  follows.  Before  testing  any  medium 
it  is  boiled  in  the  porcelain  dish  into  which  titration  takes  place. 
The  soda  solutions  are  best  stored  in  bottles  such  as  that  shown 
in  Fig.  52,  having  on  the  air  inlet  a  little  bottle  filled  with  soda 
lime  with  tubes  fitted  as  in  the  large  one.  The  CO2  of  the  air 
which  passes  through  is  thus  removed. 

Method.  —  The  practical  application  of  these  principles  is  as 
follows.  Take  the  medium  with  all  its  constituents  dissolved 
and  filter  it.  Place  5  c.c.  in  a  porcelain  dish,  add  45  c.c.  of  dis- 
tilled water  and  I  c.c.  phenol-phthaleine,  and  boil.  Run  in  deci- 
normal  soda  till  neutral  point  is  reached.  Repeat  process  thrice 
and  take  the  mean  to  obtain  amount  of  soda  required.  From 
this  calculate  acidity  of  medium  per  litre,  and  neutralise  with 
normal  soda  solution.  Check  calculation  by  a  fresh  titration  of 
5  c.c.  of  the  neutralised  medium.  Steam  for  half  an  hour,  and 
take  reaction  again  to  see  that  it  is  constant.  Now  add  normal 
HC1  in  the  ratio  of  1.5  c.c.  per  cent. 

1  For  the  majority  of  pathogenic  bacteria,  Fuller's  optimum  reaction  is  still 
too  acid.  It  will  be  found  that  an  acid  reaction  of  -5-.3  is  much  more  satis- 
factory. 


'3$  METHODS    OF   CULTIVATION   OF   BACTERIA. 

The  gelatin  and  agar  media  (vide  infra}  are  treated  in  the 
same  way. 

"  i  (b).  Glucose  Broth. — To  the  other  constituents  of  i  (a)  there 
is  added  i  or  2  per  cent  of  grape  sugar.  The  steps  in  the  prep- 
aration are  the  same.  Glucose  being  a  reducing  agent,  no  free 
oxygen  can  exist  in  a  medium  containing  it,  and  therefore  glu- 
cose broth  is  used  as  a  culture  fluid  for  anaerobic  organisms. 

1  (c\  Glycerin  Broth.  —  The  initial  steps  are  the  same  as  in 
I  (a\  but  after  filtration  6  to  8  per  cent  of  glycerin  (sp.  grav. 
1.25)  is  added.     This  medium  is  especially  used  for  growing  the 
tubercle  bacillus  when  the  soluble  products  of  the  growth  of  the 
latter  are  required. 

2.  Agar  Media  (French,  "  gelose  "  ).  —  The  disadvantage  of 
gelatin  is  that  at  the  blood  temperature  (38°  C.),  at  which  most 
pathogenic  organisms  grow  best,  it  is  liquid.  To  get  a  medium 
which  will  be  solid  at  this  temperature,  agar  is  used  as  the  stiffen- 
ing agent  instead  of  gelatin.  Unlike  the  latter,  which  is  a  proteid, 
agar  is  a  carbohydrate.  It  is  derived  from  the  stems  of  various 
seaweeds  growing  in  the  Chinese  seas,  popularly  classed  together 
as  "  Ceylon  Moss."  The  best  for  bacteriological  purposes  is 
that  consisting  of  the  thin  dried  stem  of  the  seaweed  itself. 

2  (a).  Ordinary  Agar.  —  To  preserve  as  far  as  possible  the 
relative  proportions  of  the  ingredients  throughout  the  process 
to  the  end,  it  will  be  found  most  advantageous  to  ascertain  the 
weight  of  the  finished  product  just  previous  to  filtration,  for 
then  one  can  accurately  make  up  deficiencies  due  to  evapora- 
tion, or  adjust  excesses  by  further  boiling  down.     To  this  end 
a  large  saucepan  is  taken  and  its  weight  ascertained,  and,  it 
being  required  to  make  1000  grammes  of  agar,  1500  c.c.  of  water 
are  poured  into  the  pan  and  set  over  the  flame  of  a  triple  Bunsen 
burner,  or  a  laboratory  furnace,  and  brought  to  the  boil.     At  this 
juncture  1 5  grammes  of  the  dry  agar  are  shredded  up  and  dropped 
in,  along  with  2.5  grammes  of  Liebig's  meat  extract.    The  boiling 
is  continued  until  the  agar  is  quite  dissolved,  usually  occupying 
about  thirty  minutes,  when  the  scum  which  rises  to  the  surface 
should  be  skimmed  off,  the  fire  lowered  until  boiling  ceases,  and 
then  10  grammes  of  peptone  and  5  grammes  of  common  salt  should 
be  gradually  dusted  into  the  fluid  with  constant  stirring  to  pre- 
vent the  formation  of  large  lumps  of  peptone,  the  pan  replaced 
over  the  flame  and  contents  boiled  until  all  the  peptone  is  com- 


AGAR   MEDIA.  39 

pletely  dissolved.  The  reaction  of  the  material  is  now  adjusted 
according  to  the  directions  given  under  i  (a).  Previous  to  nitra- 
tion the  medium  has  to  be  clarified,  and  to  accomplish  this  the 
pan  is  removed  from  the  fire  and  its  contents  cooled  down  to 
60°  C.,  and  to  them  are  added  the  yolks  and  whites  of  two 
eggs  beaten  up  gently  in  100  c.c.  of  water.  The  pan  is  replaced 
upon  the  fire,  and  with  a  low  burning  flame  the  heat  of  the  pan 
is  to  be  gently  and  carefully  raised  to  the  boiling-point,  so  that 
the  coagulation  of  the  eggs  may  be  produced  gradually.  After 
coagulation  is  completed,  it  is  important  to  boil  the  agar  gently 
for  about  ten  or  fifteen  minutes  to  allow  the  surface  coagulum 
to  toughen  sufficiently  so  that  it  will  not  readily  disintegrate 
when  the  agar  is  poured  into  the  funnel  containing  the  filter 
paper.  Before  filtering,  the  pan  and  contents  must  be  weighed, 
so  that,  deducting  50  grammes  for  each  egg 
and  the  weight  of  the  pan  itself,  the  net  result 
will  be  1000  grammes.  Filtration  is  now 
readily  accomplished  by  using  Swedish  filter 
paper  of  moderate  thickness  folded  and 
placed  in  a  wire  funnel,  such  as  shown  in 
Fig.  8,  to  prevent  contact  with  the  walls 
of  the  glass  funnel  into  which  it  is  placed,  FIG.  s.- wire  funnel  for 

supporting  paper  filter. 

and  moistened  thoroughly.  To  the  lower 
end  of  the  glass  funnel  is  affixed  about  10  cm.  of  rubber 
tubing  of  proper  calibre  carrying  a  pinchcock  and  a  glass 
delivery  tube  drawn  out  to  a  moderately  fine  bore.  The  appa- 
ratus is  now  set  up  on  a  stand  and  the  hot  agar  poured  carefully 
into  the  paper  filter,  the  coagulum  being  held  back  by  a  small 
oval  strainer  mounted  on  a  handle,  or  by  a  large  spoon.  The 
agar  rapidly  at  first  makes  it  way  through  the  paper  into  the 
glass  funnel,  when  it  is  at  once  delivered  into  sterile  tubes  in 
quantities  of  12  c.c.,  or  thereabouts;  later  on  because  of  cooling 
and  plugging  of  the  pores  of  the  paper  by  small  particles  of 
coagula,  the  agar  comes  through  slower  and  slower,  so  that  it- 
is  then  necessary  to  empty  it  into  the  pan  to  be  heated  up  and 
again  poured  into  a  fresh  filter ;  this  procedure  being  repeated 
as  often  as  necessary.  Filtration  may  also  be  accomplished  by 
passing  the  hot  agar  through  absorbent  cotton  packed  not  too 
tightly  into  the  neck  of  a  glass  funnel  and  wetted,  or  we  may 
have  recourse  to  the  use  of  a  hot-water  funnel  (Fig.  9). 


METHODS    OF    CULTIVATION    OF    BACTERIA. 


This  consists  of  an  outer  tin  funnel,  the  neck  of  which  is  fitted 
with  a  perforated  cork,  through  which  is  placed  the  stem  of  an 
inner  glass  funnel.  The  interspace  between 
the  two  funnels  is  filled  with  water,  which 
is  kept  hot  by  a  Bunsen  under  a  side  arm 
let  into  the  outer  funnel.  Whichever  in- 
strument be  used,  before  filtering  shake  up 
the  melted  medium,  as  it  is  apt  while  melt- 
ing to  have  settled  into  layers  of  dif- 
ferent density.  The  medium  when  tubed 
is  to  be  sterilised  in  the  autoclave  seven 
minutes. 

FIG.  9.-Hot-water funnel.          2  (*)•  Glycerin  Agar.  —  To  2  (a)  after 
filtration  add  6  to  8  per  cent  of  glycerin 

and  sterilise  as  above.     This  is  used  especially  for  growing  the 
tubercle  bacillus. 

2  (c).  Glucose  Agar.  —  Prepare  as  in  2  (a),  but  add   i  to  2 
per  cent  of  grape  sugar  along  with  agar.     This  medium  is  used 
for  the  culture  of  anaerobic  organisms  at  temperatures  above 
the   melting-point   of    gelatin.     It   is   also    a    superior    culture 
medium  for  some  aerobes,  e.g.  the  B.  diphtheriae. 

3.  Gelatin  Media.  —  These  are  simply  the  foregoing  broths, 
with  gelatin  added  as  a  solidifying  body. 

3  (a).  Peptone  Gelatin. — To   noo  c.c.  of  water  brought  to 
the  boiling-point  in  a  saucepan  of  known  weight,   100  to   150 
grammes  of  "gold  label"  gelatin  (preferably  that  of  Coignet 
et  Cie.,  Paris)  are  added,  the  solution  being  rapidly  made  by 
grasping  the  bunch  of  leaves  by  one  end  in  the   hand,  and 
stirring  it  around  in  the  water.     As  soon  as  solution  is  accom- 
plished the  fluid  is  no  longer  permitted  to  boil,  as  prolonged 
boiling  tends  to  destroy  the  gelatinising  power  of  the  medium. 
The  further  technique  is  quite  the  same  as  that  described  for 
making  agar.     The  medium  when  tubed  may  be  sterilised  for 
five  minutes  in  the  autoclave  at  120°  C,  without  endangering 
its  subsequent  gelatinisation,  if  upon  removal  from  the  autoclave 
the  tubes  are  placed  in  ice-water  until  solidified  —  a  long  experi- 
ence justifies  the  use  of  the  autoclave  under  such  conditions. 
Too  much  boiling,  or  boiling  at  too  high  a  temperature,  as  has 
been  said,  causes  a  gelatin  medium  to  lose  its  property  of  solidi- 
fication.    This  transparent  solid  gelatin  medium  is  that  chiefly 


PEPTONE   GELATIN.  4I 

employed  for  the  culture  of  aerobic  bacteria  at  ordinary  tem- 
peratures. The  exact  percentage  of  gelatin  used  in  its  prepara- 
tion depends  on  the  temperature  at  which  growth  is  to  take  place. 
Its  firmness  is  its  most  valuable  characteristic,  and  to  maintain 
this  in  summer  weather,  15  parts  per  100  are  necessary.  A 
limit  is  placed  on  higher  percentages  by  the  fact  that,  if  the 
gelatin  be  too  stiff,  it  will  split  on  the  perforation  of  its  sub- 
stance by  the  platinum  needle  used  in  inoculating  it  with  a 
bacterial  growth ;  15  per  cent  gelatine  melts  at  about  24°  C 

3  (b).  Glucose  Gelatin.  —  The  constituents  are  the  same  as 
3  (0),  with  the  addition  of  I  to  2  per  cent  of  grape  sugar.  The 
method  of  preparation  is  identical.  This  medium  is  used  for 
growing  anaerobic  organisms  at  the  ordinary  temperatures. 

3  (c).  Beerwort  Gelatin  consists  of  hopped  beerwort,  to 
which  has  been  added  10  or  15  per  cent  of  gelatin.  It  is  a  use- 
ful medium  for  the  cultivation  of  yeasts  and  moulds. 

These  bouillon,  agar,  and  gelatin  preparations  constitute  the 
most  frequently  used  media.  Growths  on  bouillon  do  not 
usually  show  any  characteristic  appearances  which  facilitate 
classification,  but  such  a  medium  is  of  great  use  in  investigating 
the  soluble  toxic  products  of  bactpria.  The  most  characteristic 
developments  of  organisms  take  place  on  the  gelatin  media. 
These  have,  however,  the  disadvantage  of  not  being  available 
when  growth  is  to  take  place  at  any  temperature  above  24°  C. 
For  higher  temperatures  agar  must  be  employed.  Agar  is,  how- 
ever, never  so  transparent.  Though  quite  clear  when  fluid,  on 
solidifying  it  always  becomes  slightly  opaque.  Further,  growths 
upon  it  are  never  so  characteristic  as  those  on  gelatin.  It  is,  for 
instance,  never  liquefied,  whereas  some  organisms,  by  their  growth, 
liquefy  gelatin  and  others  do  not  —  a  fact  of  prime  importance. 

Litmus  Media.  —  To  any  of  the  above  media  litmus  (French, 
"  tournesol ")  may  be  added  to  show  change  in  reaction  during 
bacterial  growth.  The  litmus  is  added,  before  sterilisation,  as 
a  strong  watery  solution 1  in  sufficient  quantity  to  give  the 
medium  a  distinctly  bluish  tint.  During  the  development  of  an 
acid  reaction  the  colour  changes  to  a  pink  and  may  subsequently 
be  discharged. 

Petruschky's  Litmus- whey  (as  modified  by  Durham).  —  "  Fresh 
milk  is  slightly  warmed  and  clotted  by  means  of  essence  of 

1  6  grammes  of  Merck's  neutral  extract  of  litmus  to  1000  c.c.  of  distilled  water. 


42  METHODS   OF   CULTIVATION    OF   BACTERIA. 

rennet.  The  whey  is  strained  off  and  the  clot  is  hung  up  to 
drain  in  a  piece  of  muslin.  The  whey,  which  is  somewhat 
turbid  and  yellow,  is  then  cautiously  neutralised  with  4  per  cent 
citric  acid  solution,  neutral  litmus  being  used  as  an  indicator. 
When  it  gives  a  good  neutral  violet  colour  with  the  litmus,  it  is 
heated  upon  a  water  bath  at  100°  C.  for  half  an  hour  or  so  ; 
thereby  nearly  the  whole  of  the  proteid  is  coagulated.  It  is 
then  filtered  clear,  and  neutral  litmus  is  added  to  a  convenient 
colour  for  titrations  or  rougher  observation."  It  may  be  steril- 
ised in  tubes  or  in  bulk  at  100°  C.,  or  by  passing  through  a 
Berkfeld  filter. 

Use  of  neutral  red.  —  This  dye  has  recently  been  introduced 
as  an  aid  in  determining  the  presence  or  absence  of  members 
of  the  B.  coli  group,  especially  in  the  examination  of  water. 
The  medium  found  most  suitable  is  agar  containing  .5  per  cent 
of  glucose,  to  which  i  to  5  per  cent  of  a  concentrated  solution  of 
neutral  red  is  added.  The  use  of  this  medium  and  its  probable 
value  are  described  below. 

Blood  Agar  and  Serum  Agar. — The  former  medium  was 
introduced  by  Pfeiffer  for  growing  the  influenza  bacillus,  and  it 
has  been  used  for  the  organisms  which  are  not  easily  grown  on 
the  ordinary  media,  e.g.  the  gonococcus  and  the  pneumococcus. 
Human  blood  or  the  blood  of  animals  may  be  used.  "Sloped 
tubes  "  (vide  p.  50)  of  agar  are  employed  (glycerin  agar  is  not  so 
suitable).  Purify  a  finger  first  with  i-iooo  corrosive  sublimate, 
dry,  and  then  wash  with  absolute  alcohol  to  remove  the  sublimate. 
Allow  the  alcohol  to  evaporate.  Prick  with  a  needle  sterilised 
by  heat,  and,  catching  a  drop  of  blood  in  the  loop  of  a  sterile 
platinum  wire  (vide  p.  52),  smear  it  on  the  surface  of  the  agar. 
The  excess  of  the  blood  runs  down  and  leaves  a  film  on  the 
surface.  Cover  the  tubes  with  india-rubber  caps,  and  incubate 
them  for  one  to  two  days  at  37°  C.  before  use,  to  make  certain 
that  they  are  sterile.  Agar  poured  out  in  a  thin  layer  in  a 
Petri  dish  may  be  smeared  with  blood  in  the  same  way  and 
used  for  cultures.  In  investigating  the  diseases  of  races  other 
than  the  white,  it  appears  advisable  to  use  the  blood  of  the  race 
under  investigation. 

Serum  agar  is  prepared  in  a  similar  way  by  smearing  the  sur- 
face of  the  agar  with  blood  serum,  or  by  adding  a  few  drops  of 
serum  to  the  tube  and  then  allowing  it  to  flow  over  the  surface. 


BLOOD    SERUM. 


43 


Peptone  Solution  or  DunJiams  Medium. 

A  simple  solution  of  peptone  (Witte)  constitutes  a  suitable 
culture  medium  for  many  bacteria.  The  peptone  in  the  propor- 
tion of  i  to  2  per  cent,  along  with  .5  per  cent  NaCl,  is  dissolved 
in  distilled  water  by  heating.  The  fluid  is  then  filtered,  placed 
in  tubes  and  sterilised.  The  reaction  is  usually  distinctly  alka- 
line, which  condition  is  suitable  for  most  purposes.  For  special 
purposes  the  reaction  may  be  standardised.  In  such  a  solution 
the  cholera  vibrio  grows  with  remarkable  rapidity.  It  is  also 
much  used  for  testing  the  formation  of  indol  by  a  particular 
bacterium ;  and  by  the  addition  of  one  of  the  sugars  to  it  the 
fermentative  powers  of  an  organism  may  be  tested.  Litmus 
may  be  added  to  show  any  change  in  reaction. 

Blood  Serum. 

Koch  introduced  this  medium,  and  it  is  prepared  as  follows : 
Plug  the  mouth  of  a  tall  cylindrical  glass  vessel  (say  of  1000  c.c. 
capacity)  with  cotton  wool,  and  sterilise  by  steaming  it  in  a 
Koch's  steriliser  for  one  and  a  half  hours.  Take  it  to  the  place 
where  a  horse,  ox,  or  sheep  is  to  be  killed.  When  the  artery  or 
vein  of  the  animal  is  opened,  allow  the  first  blood  which  flows, 
and  which  may  be  contaminated  from  the  hair,  etc.,  to  escape ; 
fill  the  vessel  with  the  blood  subsequently  shed.  Carry  care- 
fully back  to  the  laboratory  without  shaking,  and  place  for 
twenty-four  hours  in  a  cool  place,  preferably  an  ice-chest.  The 
•clear  serum  will  separate  from  the  clotted  blood.  If  a  centrifuge 
is  available,  a  large  yield  of  serum  may  be  obtained  by  centrifu- 
galising  the  freshly  drawn  blood.  If  coagulation  has  occurred, 
the  clot  must  first  be  thoroughly  broken  up.  With  a  sterile 
10  c.c.  pipette  transfer  this  quantity  of  serum  to  each  of  a  series 
•of  test-tubes  which  must  previously  have  been  sterilised  by  dry 
heat.  The  serum  may,  with  all  precautions,  have  been  con- 
taminated during  the  manipulations,  and  must  be  sterilised. 
As  it  will  coagulate  if  heated  above  68°  C,  advantage  must  be 
taken  of  the  intermittent  process  of  sterilisation  at  57°  C. 
[method  B  (4)].  It  is  therefore  kept  for  one  hour  at  this 
temperature  on  each  of  eight  successive  days.  It  is  always 
well  to  incubate  it  for  a  day  at  37°  C.  before  use,  to  see  that 
the  result  is  successful.  After  sterilisation  it  is  "  inspissated," 


44 


METHODS    OF   CULTIVATION   OF   BACTERIA. 


FlG.  10.  —  Blood  serum  inspissator. 


by  which  process  a  clear  solid  medium  is  obtained.  "  Inspissa- 
tion  "  is  probably  an  initial  stage  of  coagulation,  and  is  effected 
by  keeping  the  serum  at  65°  C.  till  it  stiffens.  This  temperature 
is  just  below  the  coagulation  point  of  the  serum.  The  more 
slowly  the  operation  is  performed  the  clearer  will  be  the  serum. 
The  apparatus  used  is  seen  in  Fig.  10.  It  consists  of  a  rectan- 
gular, shallow,  covered,  hot-water 
jacket,  with  sloped  bottom,  and  can 
be  rapidly  heated  by  an  S-shaped 
Bunsen  containing  many  lateral 
perforations,  from  each  of  which 
a  flame  issues.  The  serum  tubes 
are  thus  placed  in  a  sloped  posi- 
tion, and  the  temperature  being 
raised  to  65°  C.,  the  contents 
solidify  in  a  sloped  position  in  the 
interior.  It  is  well  not  only  to 
have  the  jacket  filled  with  water, 
but  also  to  put  some  water  in  the 

trough  in  which  the  tubes  lie,  and  also  to  have  a  thermometer  in  the 
water.  This  prevents  cooling  of  the  tubes  when  the  lid  is  raised 
to  see  if  the  process  is  complete.  As  is  evident,  the  prepara- 
tion of  this  medium  is  tedious,  but  its  use  is  necessary  for  the 
observation  of  particular  characteristics  in  several  pathogenic 
bacteria,  notably  the  tubercle  bacillus.  Pleuritic  and  other 
effusions  may  be  prepared  in  the  same  way,  and  used  as  media, 
but  care  must  be  taken  in  their  use,  as  we  have  no  right  to  say 
that  pathological  effusions  have  the  same  chemical  composition 
as  normal  serum. 

If  blood  be  collected  with  strict  aseptic  precautions,  then 
sterilisation  of  the  serum  is  unnecessary.  To  this  end  the  mouth 
of  the  cylinder  used  for  collecting  the  blood,  instead  of  being 
plugged  with  wool,  has  an  india-rubber  bung  inserted  in  it 
through  which  two  bent  glass  tubes  pass.  The  outer  end  of  one 
of  these  is  of  convenient  length,  and,  before  sterilisation,  a  large 
cap  of  cotton  wool  is  tied  over  it;  the  other  tube  is  plugged 
with  a  piece  of  cotton  wool.  In  the  slaughter-house  the  cap  is 
removed  and  the  tube  is  inserted  into  the  blood-vessel  as  a 
canula.  The  cylinder  is  thus  easily  filled.  Another  method  is 
to  conduct  the  blood  to  the  cylinder  by  means  of  a  sterilised 


SERUM    MEDIA.  45 

canula  and  india-rubber  tube,  the  former  being  inserted  in  the 
blood-vessel.  The  serum  obtained  under  such  circumstances 
must  be  incubated  before  use,  to  make  sure  that  it  is  sterile. 

Lbffler's  Blood  Serum.  —  This  is  the  best  medium  for  the 
growth  of  the  B.  diphtheriae  and  may  be  used  for  other  organisms. 
It  has  the  following  composition.  Three  parts  of  calf's  or  lamb's 
blood  serum  are  mixed  with  one  part  ordinary  neutral  peptone 
bouillon  made  from  veal  with  I  per  cent  of  grape  sugar  added  to 
it.  Though  this  is  the  original  formula  it  can  be  made  from  ox 
or  sheep  serum  and  beef  bouillon  without  its  qualities  being 
markedly  impaired.  Sterilise  by  method  B  (4)  as  above  (p.  32). 
Or  we  may  adopt  the  more  rapid  method  in  bringing  about 
coagulation  at  a  hf£h  temperature,  by  placing  the  tubes  as  before 
in  the  inspissator  and  slowly  raising  the  water  in  the  jacket  of 
the  apparatus  to  the  boiling-point  and  then  turning  out  the  gas. 
The  serum  will  be  found  to  be  firmly  coagulated  and  free  from 
bubbles,  if  the  precaution  were  taken  to  keep  the  tubes  of 
serum  free  from  contact  with  the  metal  bottom  or  sides  of  the 
inspissator  by  means  of  thin  wooden  slats  properly  adjusted. 
The  medium  must  then  be  sterilised  on  three  successive  days  in 
the  Koch  or  Arnold  steriliser. 

Alkaline  Blood  Serum  (Lorrain  Smith's  Method).  —  To  each 
100  c.c.  of  the  serum  obtained  as  before,  add  i  to  1.5  c.c.  of  a 
10  per  cent  solution  of  sodium  hydrate  and  shake  it  gently. 
Put  sufficient  of  the  mixture  into  each  of  a  series  of  test-tubes, 
and  laying  them  on  their  sides,  sterilise  by  method  B  (2).  If  the 
process  of  sterilisation  be  carried  out  too  quickly,  bubbles  of  gas 
are  apt  to  form  before  the  serum  is  solid,  and  these  interfere 
with  the  usefulness  of  the  medium.  Dr.  Smith  informs  us  that 
this  can  be  obviated  if  the  serum  be  solidified  high  up  in  the 
Koch's  steriliser,  in  which  the  water  is  allowed  only  to  simmer. 
In  this  case  sterilisation  ought  to  go  on  for  one  and  a  half  hours. 
A  clear  solid  medium  (consisting  practically  of  alkali-albumin)  is 
thus  obtained,  and  he  has  found  it  of  value  for  the  growth  of  the 
organisms  for  which  Koch's  serum  is  used,  and  especially  for 
the  growth  of  the  B.  diphtheriae.  Its  great  advantage  is  that 
aseptic  precautions  in  obtaining  blood  from  the  animal  are  not 
necessary,  and  it  is  easily  sterilised. 

Marmorek's  Serum  Media.  —  There  has  always  been  a  diffi- 
culty in  maintaining  the  virulence  of  cultures  of  the  pyogenic 


46  METHODS   OF   CULTIVATION   OF   BACTERIA. 

streptococci,  but  Marmorek  has  succeeded  in  doing  so  by  grow- 
ing them  on  the  following  media,  which  are  arranged  in  the 
order  of  their  utility  :  — 

1.  Human  serum  2  parts,  bouillon  I  part. 

2.  Pleuritic  or  ascitic  serum  I  part,  bouillon  2  parts. 

3.  Asses'  or.mules'  serum  2  parts,  bouillon  i  part. 

4.  Horse  serum  2  parts,  bouillon  i  part. 

Human  serum  can  be  obtained  from  the  blood  shed  in 
venesection,  the  same  precautions  being  taken  as  in  the  case  of 
that  got  in  the  slaughter-house.  In  the  case  of  these  media, 
sterilisation  is  effected  by  method  B  (4),  and  they  are  used  fluid. 

Hiss's  Serum  Media.  —  Their  use  is  for  a  more  efficient 
means  of  differentiating  pneumococcus  from  streptococcus  pyo- 
genes,  and  they  bear  some  resemblance  to  those  of  Marmorek. 

A.  Ox  serum  i  part. 
Distilled  water  2  parts. 

Normal  sodium  hydrate  o.  i  per  cent. 

B.  Ox  serum  i  part. 
Distilled  water  2  parts. 
Inulin  i.o  per  cent. 

They  can  be  sterilised  intermittently  at  100°  C.  without 
coagulating.  In  either  medium  pneumococcus  forms  acid  and 
coagulates  the  serum,  but  more  rapidly  in  the  latter,  whilst 
streptococcus  does  neither. 

Potatoes  as  Culture  Material. 

(a)  In  Potato  Jars.  —  The  jar  consists  of  a  round,  shallow, 
glass  vessel  with  a  similar  cover  (vide  Fig.  1 1 ).  It  is  washed 

with  i-iooo  corrosive  sublimate,  and  a 
piece  of  circular  filter  paper,  moistened 
with  the  same,  is  laid  in  its  bottom.  On 
this  latter  are  placed  four  sterile  watch- 
glasses.  Two  firm,  healthy,  small, 
round  potatoes,  as  free  from  eyes  as 

FIG.  ii. -Potato  jar.  possible,  and  with  the  skin  whole,  are 

scrubbed  well  with  a  brush  under  the  tap 

and  steeped  for  two  or  three  hours  in  i-iooo  corrosive  sublimate. 
They  are  steamed  in  the  Koch's  steriliser  for  thirty  minutes  or 


POTATO    AS   A   CULTURE    MEDIUM. 


47 


FIG.  12.  — Cylinder  of 
potato  cut  obliquely. 


longer,  or  in  the  autoclave  for  a  quarter  of  an  hour.  When 
cold,  each  is  grasped  between  the  left  thumb  and  forefinger 
(which  have  been  sterilised  with  sublimate)  and  cut  through  the 
middle  with  a  sterile  knife.  It  is  best  to  have  the  cover  of  the 
jar  raised  by  an  assistant,  and  to  perform  the  cutting  beneath  it. 
Each  half  is  put  in  one  of  the  watch-glasses,  the  cut  surfaces, 
which  are  then  ready  for  inoculation  with  a  bacterial  growth, 
being  uppermost.  Smaller  jars,  each  of  which  holds  half  of  a 
potato,  are  also  used  in  the  same  way  and  are  very  convenient. 

(b)  By  Slices  in  Tubes. — This  method,  introduced  by  Ehrlich, 
is  the  best  means  of  utilising  potatoes  as  a  medium.  A  large, 
long  potato  is  well  washed  and  scrubbed, 
and  peeled  with  a  clean  knife.  A  cylinder 
is  then  bored  from  its  interior  with  an 
apple  corer  or  a  large  cork  borer,  and  is 
cut  obliquely,  as  in  Fig.  12.  Two  wedges 
are  thus  obtained,  and  to  preserve  their 
white  appearance  as  much  as  possible  these  slices  are  placed 
in  running  water  for  12  to  18  hours,  then  deposited  broad  end 
down  in  a  test-tube  of  special  form  (see  Fig.  13).  In  the  wide 
part  at  the  bottom  of  this  tube  is  placed  a  piece  of  cotton 
wool,  which  catches  any  condensation  water  which  may  form. 
The  wedge  rests  on  the  constriction  above  this 
bulbous  portion.  The  tubes,  washed,  dried,  and 
with  cotton  wool  in  the  bottom  and  in  the  mouth, 
are  sterilised  before  the  slices  of  potato  are  intro- 
duced. After  the  latter  are  inserted,  the  tubes  are 
sterilised  in  the  Koch  steam  steriliser  for  one  hour, 
or  in  the  autoclave  for  five  minutes  at  I  atmos- 
phere pressure  and  120°  C.  An  ordinary  test-tube 
may  be  used  with  a  piece  of  sterile  absorbent  wool 
in  its  bottom,  on  which  the  potato  may  rest. 

Glycerin  potato,  suitable  for  the  growth  of  the 
tubercle   bacillus,  may  be    prepared   by  covering 
the  slices  in  the  tubes  with  6  per  cent  solution  of 
-  gtycerin  in  water  and  steaming  for  half  an  hour, 
taming  piece  of  The  fluid  is  then  poured.off  and  the  sterilisation 

continued  for  another  half-hour. 

Potatoes  ought  not  to  be  prepared  long  before  being  used, 
as  the  surface  is  apt  to  become  dry  and  discoloured.     It  is  well 


48  METHODS   OF   CULTIVATION   OF   BACTERIA. 

to  take  the  reaction  of  the  potato  with  litmus  before  sterilisation, 
as  this  varies  ;  normally  in  young  potatoes  it  is  weakly  acid. 
The  reaction  of  the  potato  may  be  more  accurately  estimated  by 
steaming  the  potato  slices  for  a  quarter  of  an  hour  in  a  known 
quantity  of  distilled  water  and  then  estimating  the  reaction  of 
the  water  by  phenol-phthaleine.  The  required  degree  of  acidity 
or  alkalinity  is  obtained  by  adding  the  necessary  quantity  of 
HC1  or  NaOH  solution  (p.  37)  and  steaming  for  other  fifteen 
m;nutes.  The  water  is  then  poured  off  and  sterilisation  con- 
tinued for  another  half-hour.  Potatoes  before  being  inoculated 
ought  always  to  be  incubated  at  37°  C.  for  a  night,  to  make  sure 
that  their  sterilisation  has  been  successful.  This  is  necessary 
only  if  sterilised  in  the  Koch  steamer. 

Eisner's  Medium.  —  This  is  one  of  the  media  introduced  in  the  study  of 
the  comparative  reactions  of  the  typhoid  bacillus  and  the  bacillus  coli.  The 
preparation  is  as  follows  :  500  grammes  potato  are  grated  up  in  a  litre  of  water, 
allowed  to  stand  over  night,  then  strained,  and  added  to  an  equal  quantity  of 
ordinary  15  per  cent  peptone  gelatin  which  has  not  been  neutralised.  Normal 
sodium  hydrate  solution  is  added  till  the  reaction  is  feebly  acid,  the  whole 
boiled  together,  filtered,  and  sterilised.  Just  before  use  potassium  iodide  is 
added  so  as  to  constitute  one  per  cent  of  the  amount  used.  Moore  has  used 
a  similar  agar  preparation.  Here  500  grammes  potato  are  scraped  up  in  one 
litre  of  water,  allowed  to  stand  for  three  hours,  strained,  and  put  aside  over 
night.  The  clear  fluid  is  poured  off,  made  up  to  one  litre,  rendered  slightly 
alkaline,  20  grammes  agar  are  added,  and  the  whole  is  treated  as  in  making 
ordinary  agar.  The  medium  is  distributed  in  test-tubes — 10  c.c.  to  each  — 
and  immediately  before  use,  to  each  is  added  .5  c.c.  of  a  solution  of  10  grammes 
potassium  iodide  to  50  c.c.  water. 

Milk  as  a  Ctilture  Medium. 

This  is  a  convenient  medium  for  observing  the  effects  of 
bacterial  growth  in  changing  the  reaction,  in  coagulating  the 
soluble  albumin,  and  in  fermenting  the  lactose.  It  is  prepared 
as  follows  :  fresh  milk  is  taken,  preferably  after  having  had  the 
cream  "separated"  by  centrifugalisation,  as  is  practised  in  the 
best  dairies,  and  is  steamed  for  fifteen  minutes  in  the  Koch ;  it  is 
then  set  aside  in  an  ice-chest  or  cool  place  over  night  to  facilitate 
further  separation  of  cream.  The  milk  is  siphoned  off  from 
beneath  the  cream.  The  reaction  of  fresh  milk  is  alkaline.  If 
great  accuracy  is  necessary,  any  required  degree  of  reaction  may 
be  obtained  by  the  titration  method.  It  is  then  placed  in  tubes 
and  sterilised  by  methods  B  (2)  or  B  (3). 


THE   USE   OF   CULTURE  MEDIA.  49 

Added  value  is  given  to  the  usefulness  of  milk  by  the  addi- 
tion to  each  litre  of  150  c.c.  of  a  watery  tincture  of  litmus 
(Merck's  extract  of  litmus,  6  grammes;  water,  1000  c.c.),  whilst 
the  retention  of  a  small  percentage  of  cream  confers  a  distinct 
advantage. 

Bread  Paste. 

This  is  useful  for  growing  torulae,  moulds,  etc.  Some  ordi- 
nary bread  is  cut  into  slices,  and  then  dried  in  an  oven  till  it  is 
so  dry  that  it  can  be  pounded  to  a  fine  powder  in  a  mortar,  or 
rubbed  down  with  the  fingers  and  passed  through  a  sieve.  Some 
100  c.c.  flasks  are  washed,  dried,  and  sterilised,  and  a  layer  of 
the  powder,  half  an  inch  thick,  placed  on  the  bottom.  Distilled 
water,  sufficient  to  cover  the  whole  of  it,  is  then  run  in  with  a 
pipette  held  close  to  the  surface  of  the  bread,  and,  the  cotton- 
wool plugs  being  replaced,  the  flasks  are  sterilised  in  the  Koch's 
steriliser  by  method  B  (2).  The  reaction  is  slightly  acid. 

Beerwort. 

Beerwort,  and  beerwort  to  which  10  per  cent  of  gelatin  has 
been  added,  are  also  used  for  the  study  of  these  higher  organ- 
isms. One  takes  ordinary  hopped  beerwort  and  places  it  in,  say, 
a  litre  flask,  autoclaves  it  for  five  minutes,  cools  it  thoroughly,  and 
filters ;  by  this  means  all  resinous  matters  are  precipitated  and 
removed,  and  the  wort  remains  permanently  clear.  It  is  tubed, 
and  sterilised  in  the  autoclave  for  seven  minutes,  or,  as  usual,  in 
the  Koch  or  Arnold  apparatus. 

THE  USE  OF  THE  CULTURE  MEDIA. 

The  culture  of  bacteria  is  usually  carried  on  in  test-tubes 
conveniently  6  x  f  in.  If  new,  these  ought  to  be  carefully 
washed  and  dripped,  and  their  mouths  are  plugged  with  pledgets 
of  plain  cotton  wool.  They  are  then  sterilized  for  one  hour  at 
170°  C.  The  reason  is  that  the  glass,  being  usually  packed  in 
straw,  is  covered  with  the  extremely  resisting  spores  of  the  ba- 
cillus subtilis.  Tubes  which  have  been  in  use  are  merely  well 
washed,  dried  thoroughly,  and  plugged.  Cotton-wool  plugs  are 
universally  used  for  protecting  the  sterile  contents  of  flasks 
and  tubes  from  contamination  with  the  bacteria  of  the  air.  A 
medium  thus  protected  will  remain  sterile  for  years.  Whenever 
a  protecting  plug  is  removed  for  even  a  short  time,  the  sterility 


METHODS    OF   CULTIVATION    OF   BACTERIA. 


of  the  contents  may  be  endangered.     It  is  well  to  place  the 

bouillon,  gelatin,  and  agar  media  in 
the  test-tubes  directly  after  filtration. 
The  media  can  then  be  sterilized  in 
the  test-tubes. 

In  filling  tubes,  care  must  be 
taken  to  run  the  liquid  down  the 
centre,  so  that  none  of  it  drops  on 
the  inside  of  the  upper  part  of  the 
tube  with  which  the  cotton-wool 
plug  will  be  in  contact,  otherwise 
the  latter  will  subsequently  stick  to 
the  glass  and  its  removal  will  be 
difficult.  The  tubes  may,  when 
filled,  be  placed  in  cages  made  of 
fine  wire  netting  and  sterilised.  If 
all  the  contents  of  a  flask  of  medium 
be  not  filled  into  tubes,  the  remain- 
FIG.  14. — Apparatus  for  delivering  der  must  be  re-sterilised  before 

measured  quantities    of   media   into    bei          stored        j      ^  f    }.{d 

tubes. 

media,    test-tubes    are    filled    about 

one-third  full.  With  solid  media  the  amount  varies.  In 
the  case  of  gelatin  media,  tubes 
filled  one-third  full  and  al- 
lowed to  solidify  while  standing 
upright  are  those  commonly  used. 
With  organisms  needing  an  abun- 
dant supply  of  oxygen  the  best 
growth  takes  place  on  the  surface 
of  the  medium,  and  for  practical 
purposes  the  surface  ought  thus 
to  be  as  large  as  possible.  To 
this  end  "sloped"  agar  and  gela- 
tin tubes  are  used.  To  prepare 
these,  tubes  are  filled  only  about 
one-sixth  full,  and  after  sterilisa- 
tion are  allowed  to  solidify,  lying 
on  their  sides  with  their  necks 
supported  so  that  the  contents 
extend  3  to  4  inches  up,  giving  an  oblique  surface  when  held 


O.  f>  C 

FlG.  15.  —  Tubes  of  media. 
a  Ordinary  upright  tube.      b.   Sloped  tube. 
c.  "  Deep  "  tube  for  cultures  of  anaerobes. 


THE   USE   OF   CULTURE    MEDIA.  51 

upright  after  solidification.  Thus  agar  is  commonly  used  in 
such  tubes  (less  frequently  gelatin  is  also  "  sloped  "),  and  this 
is  the  position  in  which  blood  serum  is  inspissated.  Tubes, 
especially  those  of  the  less  commonly  used  media,  should  be 
placed  in  large  jars  provided  with  stoppers,  otherwise  the 
contents  are  apt  to  evaporate.  A  tube  of  medium  which  has 
been  inoculated  with  a  bacterium,  and  on  which  growth  has 
taken  place,  is  called  a  "culture."  A  "pure  culture"  is  one 
in  which  only  one  organism  is  present.  The  methods  of 
obtaining  pure  cultures  will  presently  be  described.  When  a 
fresh  tube  of  medium  is  inoculated  from  an  already  existing 
culture,  the  resulting  growth  is  said  to  be  a  "sub-culture"  of 


FIG.  16. —  Platinum  wires  in  glass  handles. 

a.  Straight  needle  for  ordinary  puncture  inoculations,     b.  "  Platinum  loop."    c.  Long  needle  for 
inoculating  "deep"  tubes. 

the  first.  All  manipulations  involving  the  transference  of 
small  portions  of  growth  either  from  one  medium  to  another, 
as  in  the  inoculation  of  tubes,  or,  as  will  be  seen  later,  to 
cover-glasses  for  microscopic  examination,  are  effected  by 
pieces  of  platinum  wire  (Nos.  24  or  27  Birmingham  wire 
gauge — the  former  being  the  thicker)  fixed  in  glass  rods 
8  inches  long.1  Every  worker  should  have  three  such  wires. 
Two  are  2\  inches  long,  one  of  these  being  straight  (Fig.  16,  a\ 
and  the  other  having  a  loop  turned  upon  it  (Fig.  16,  b).  The 
latter  is  referred  to  as  the  platinum  "  loop  "  or  platinum  "  eyelet," 
and  is  used  for  many  purposes.  "  Taking  a  loopful "  is  a  phrase 
constantly  used.  The  third  wire  (Fig.  16,  c)  ought  to  be  4^  inches 
long,  and  straight  It  is  used  for  making  anaerobic  cultures. 
Cultures  on  a  solid  medium  are  referred  to  (i)  as  "puncture" 
or  "stab"  cultures  (German,  Stichkultur),  or  (2)  as  "stroke" 
cultures  (Strichkultur),  according  as  they  are  made  (i)  on  tubes 
solidified  in  the  upright  position,  or  (2)  on  sloped  tubes. 

1  Aluminium  rods  are  made  which  are  very  convenient.     The  end  is  split  with  a 
knife,  the  platinum  wire  is  inserted  and  fixed  by  pinching  the  aluminium  on  it  in  a  vice^ 


52  METHODS   OF   CULTIVATION    OF   BACTERIA. 

To  inoculate,  say,  one  ordinary  upright  gelatin  tube  from 
another,  the*  two  tubes  are  held  in  an  inverted  position  between 
the  forefinger  and  thumb  of  the  left  hand,  with  their  mouths 
towards  the  person  holding  them  ;  the  plugs  are  twisted  round 
once  or  twice,  to  make  sure  they  are  not  adhering  to  the  glass. 
The  short,  straight  platinum  wire  is  then  heated  to  redness  from 
point  to  insertion,  and  2  to  3  inches  of  the  glass  rod  are  also 
passed  two  or  three  times  through  the  Bunsen  flame.  It  is  held 
between  the  right  fore  and  middle  fingers,  with  the  needle  pro- 
jecting backwards,  i.e.  away  from  the  right  palm.  Remove  plug 
from  culture  with  right  forefinger  and  thumb,  and  continue  to 
hold  it  between  the  same  fingers,  by  the  part  which  projected 
beyond  the  mouth  of  the  tube.  Now  touch  the  culture  with  the 
platinum  needle,  and,  withdrawing  it,  replace  plug.  In  the 
same  way  remove  plug  from  tube  to  be  inoculated,  and  plunge 
platinum  wire  down  the  centre  of  the  gelatin  to  within  half  an 
inch  of  the  bottom.  It  must  on  no  account  touch  the  glass  above 

the  medium.  The  wire  is 
then  immediately  sterilised. 
A  variation  in  detail  of  this 
method  is  to  hold  the  plug  of 
the  tube  next  the  thumb  be- 
tween the  fore  and  middle 
fingers,  and  the  plug  of  the 
other  between  the  middle' 
and  ring  fingers,  then  to 
make  the  inoculation  (Fig. 

FIG.  17. — Another  method  of  inoculating  1 7).          The      Sub-Cllltlire      is 

labelled,  and  in  a  bacterio- 
logical laboratory  a  label  should  never  be  licked.  If  a  tube 
contain  a  liquid  medium,  it  must  be  held  in  a  sloping  position 
between  the  same  fingers,  as  above.  When  a  stroke  culture  is 
made,  the  same  manipulations  are  gone  through.  Here  the  pla- 
tinum loop  is  used,  and  a  little  of  the  culture  is  smeared  in  a 
line  along  the  surface  of  the  medium  from  below  upwards.  In 
inoculating  tubes,  it  is  always  well,  on  removing  the  plugs,  to 
make  sure  that  no  strands  of  cotton  fibre  are  adhering  to  the 
inside  of  the  necks.  As  these  might  be  touched  with  the 
charged  needle  and  the  plug  thus  be  contaminated,  they  must 
be  removed  by  heating  the  inoculating  needle  red-hot  and 


THE    SEPARATION    OF   AEROBIC   BACTERIA. 


53 


FIG.  18.  —  Rack  for  platinum  needles. 


scorching  them  off  with  it.  When  the  platinum  wires  are  not  in 
use  they  may  be  laid  in  a  rack  made  by  bending  up  the  ends  of  a 
piece  of  tin  (as  in  Fig.  18).  To 
prevent  contamination  of  cultures 
by  bacteria  falling  on  the  plugs 
while  these  are  exposed  to  the  air 
during  inoculation  manipulations, 
some  bacteriologists  singe  the 
plugs  in  the  flame  before  replac- 
ing. This  is,  however,  in  most 
cases,  a  needless  precaution.  If  the  top  of  a  plug  be  dusty,  it 
is  best  to  singe  it  before  extraction. 

THE  METHODS  OF  THE   SEPARATION  OF  AEROBIC   ORGANISMS. 

Plate-cultures. 

The  general  principle  underlying  the  methods  of  separa- 
tion is  the  distribution  of  the  bacteria  in  one  of  the  solid  media 
liquefied  by  heat  and  the  dilution  of  the  mixture  so  that  the 
growths  produced  by  the  individual  bacteria  —  called  colonies  — 
shall  be  suitably  apart.  In  order  to  render  the  colonies  easily 
accessible,  the  medium  is  made  to  solidify  in  as  thin  a  layer  as 
possible,  by  being  poured  out  on  glass  plates  —  hence  the  term 
"plate-cultures,"  —  or  in  Petri's  dishes. 

As  the  optimum  temperature  varies  with  different  bacteria, 
it  is  necessary  to  use  both  gelatin  and  agar  media.  Many 
pathogenic  organisms,  e.g.  pneumococcus,  B.  diphtheriae,  etc., 
grow  too  slowly  on  gelatin  to  allow  its  ready  use.  On  the  other 
hand,  many  organisms,  e.g.  some  occurring  in  water,  do  not 
develop  on  agar  incubated  at  37°C. 

Separation  by  Gelatin  Media.  —  As  the  naked-eye  and  micro- 
scopic appearances  of  colonies  are  often  very  characteristic, 
plate-cultures,  besides  use  in  separa- 
tion, are  often  taken  advantage  of  in 
the  description  of  individual  organisms. 
The  plate-culture  method  can  also  be 
used  to  test  whether  a  tube-culture  is 
or  is  not  pure.  The  suspected  culture 
is  plated  (three  plates  being  prepared, 
as  will  be  described).  If  all  the  colo- 
nies are  the  same,  then  the  cultures  may  be  held  to  be  pure. 


FIG.  19.  —  Petri's  capsule  or 
dish.  (Cover  shown  partially 
raised.) 


54  METHODS    OF   CULTIVATION    OF    BACTERIA. 

Either  simple  plates  of  glass  4  inches  by  3  inches  are  used, 
or,  what  are  more  convenient,  circular  glass  cells  with  similar 
•overlapping  covers.  The  latter  are  known  as  Petri's  dishes  or 
capsules  (Fig.  19).  They  are  usually  3  inches  in  diameter  and 
half  an  inch  deep.  The  advantage  of  these  is  that  they  do  not 
require  to  be  kept  level  by  a  special  apparatus  while  the  medium 
is  solidifying,  and  can  be  readily  handled  afterwards  without 
admitting  impurities.  Whether  plates  or  Petri  dishes  are  used, 
they  are  washed,  dried  with  a  clean  cloth,  and  sterilised  for  one 
hour  in  dry  air  at  170°  C.,  the  plates  being  packed  in  sheet-iron 
boxes  made  for  the  purpose  (see  Fig.  20). 

i.  Petri's  Dishes.  — While  in  certain  circumstances,  as  when 
the  number  of  colonies  has  to  be  counted,  it  is  best  to  use  plates 
•of  glass,  in  the  usual  laboratory  routine  Petri's  dishes  are  to  be 
preferred  for  the  above  reasons. 

The  contents  of  three  gelatin  tubes,  marked  a,  b,  c,1  are 
liquefied  by  placing  in  a  beaker  of  water  at  any  temperature 
between  25°  C.  and  38°  C.  Inoculate  a  with  the  bacterial 
mixture.  The  amount  of  the  latter  to  be  taken  varies,  and  can 
only  be  regulated  by  experience.  If  the  microscope  shows 
enormous  numbers  of  different  kinds  of  bacteria  present,  just 
as  much  as  adheres  to  the  point  of  a  straight  platinum  needle 
is  sufficient.  If  the  number  of  bacilli  is  small,  one  to  three 
loops  of  the  mixture  may  be  transferred  to  the  medium.  Shake 
a  well,  but  not  so  as  to  cause  many  fine  air-bubbles  to  form. 
Transfer  two  loops  of  gelatin  from  a  to  b.  Shake  b  and  trans- 
fer two  loops  to  c.  The  plugs  of  the  tubes  are  in  each  case  re- 
placed and  the  tubes  returned  to  the  beaker.  The  contents  of 
the  three  tubes  are  then  poured  out  into  three  dishes.  In  doing 
so  the  plug  of  each  tube  is  removed  and  the  mouth  of  the  tube 
passed  two  or  three  times  through  the  Bunsen  flame,  the  tube 
being  meantime  rotated  round  a  longitudinal  axis.  Any  organ- 
isms on  its  rim  are  thus  killed.  The  dishes  are  labelled  and 
set  aside  till  growth  takes  place. 

The  colonies  appear  as  minute  rounded  points,  whitish  or 
variously  coloured.  Their  characters  can  be  more  minutely 
studied  by  means  of  a  hand-lens  or  by  inverting  the  dish  on 
the  stage  of  a  microscope  and  examining  with  a  low  power 

1  For  marking  glass  vessels  it  is  convenient  to  use  the  red,  blue,  or  yellow  wax 
pencils  made  for  the  purpose  by  Faber. 


KOCH'S   METHOD   OF  PREPARING   PLATES. 


55 


through  the  bottom.  From  their  characters,  colour,  shape, 
contour,  appearance  of  surface,  liquefaction  or  non-liquefaction 
of  the  gelatin,  etc.,  the  colonies  can  be  classified  into  groups. 
Further  aid  in  the  grouping  of  the  varieties  is  obtained  by 
making  film  preparations  and  examining  them  microscopically. 
Gelatin  or  agar  tubes  may  then  be  inoculated  from  a  colony  of 
each  variety,  and  the  growths  obtained  are  then  examined  both 
as  to  their  purity  and  as  to  their  special  characters,  with  a  view 
to  their  identification  (p.  112). 

2.  Glass  Plates  (Koch).  —  When  plates  of  glass  are  to  be 
used,  an  apparatus  on  which  they  may  be  kept  level  while 
the  medium  is  solidifying, 
is,  as  has  been  said, 
necessary.  An  apparatus 
devised  by  Koch  is  used 
(Figs.  20,  21).  This  con- 
sists of  a  circular  plate  of 
glass  (with  the  upper  sur- 
face ground,  the  lower 
polished)  on  which  the 
plate  used  for  pouring  out 
the  medium  is  placed. 
The  latter  is  protected 
from  the  air  during  solidi- 
fication by  a  bell-jar.  The  bell-jar,  where  it  subsequently  has  the  medium 
,  ,  ,  j  ,  n  .  poured  out  upon  it. 

circular  plate  and  bell-jar 

rest  on  the  flat  rim  of  a  circular  glass  trough,  which  is  filled  quite 
full  with  a  mixture  of  ice  and  water,  to  facilitate  the  lowering 
of  the  temperature  of  whatever  is  placed  beneath  the  bell-jar. 
The  glass  trough  rests  on  corks  on  the  bottom  of  a  large  circu- 
lar trough,  which  catches  any  water  that  may  be  spilled.  This 
trough  in  turn  rests  on  a  wooden  triangle  with  a  foot  at  each 
corner,  the  height  of  which  can  be  adjusted,  and  which  thus 
constitutes  the  levelling  apparatus.  A  spirit  level  is  placed 
where  the  plate  is  to  go,  and  the  level  of  the  ground-glass  plate 
thus  assured.  There  is  also  prepared  a  "damp  chamber,"  in 
which  the  plates  are  to  be  stored  after  being  made.  This  con- 
sists of  a  circular  glass  trough  with  a  similar  cover.  It  is  ster- 
ilised by  being  washed  outside  and  inside  with  perchloride  of 
mercury  i-iooo,  and  a  circle  of  filter  paper  moistened  with  the 


FIG.  20.  —  Koch's  levelling  apparatus  for  use  in 
preparing  plates.  Hands  shown  in  first  position  for 
transferring  sterile  plate  from  iron  box  to  beneath 


METHODS    OF   CULTIVATION    OF   BACTERIA. 


same  is  laid  on  its  bottom.     Glass  benches  on  which  the  plates 
may  be  laid  are  similarly  purified. 

To  separate  organisms  by  this  method  three  tubes,  a,  b,  c, 
are  inoculated  as  in  using  Petri's  dishes.  The  hands  having 
been  washed  in  perchloride  of 
mercury  i-iooo  and  dried,  the 
plate-box  is  opened,  and  a  plate 
lifted  by  its  opposite  edges 
and  transferred  to  the  levelled 
ground  glass(as  in  Figs.  20,  2 1 ). 
The  bell-jar  of  the  leveller  be- 
ing now  lifted  a  little,  the  gelatin 
in  tube  a  is  poured  out  on  the 
surface  of  the  sterile  plate,  and 
while  still  fluid  is  spread  by 
stroking  with  the  rim  of  the 
tube.  After  the  medium  solidi- 
fies, the  plate  is  transferred  to 

the    moist    Chamber    as    rapidly      which  was  undermost  in  the  latter  is  upper 

as  possible,  so  as  to  avoid  atmos- 
pheric contamination.      In  do- 

*n&  ^is,  it  is  advisable  to  have  an  assistant  to  raist 
the  glass  covers.  Tubes  b  and  c  are  similarly  treated, 
and  the  resulting  plates  stacked  in  series  on  the  top 
of  a.  The  chamber  is  labelled  and  set  aside  for  a 
few  days  till  the  colonies  appear  in  the  gelatin  plates. 
The  further  procedure  is  of  the  same  nature  as  with 
Petri's  dishes. 

3.  Esmarctis  Roll  Tubes.  —  Here  the  principle 
is  that  of  dilution  as  before.  In  each  of  three  test- 
tubes  i^  or  \\  inch  in  diameter,  gelatin  to  the  depth 
of  \  of  an  inch  is  placed.  These  are  sterilised.  The 
gelatin  is  melted  and  inoculated  in  series  with  the 
bacterial  mixture  as  in  making  plate-cultures,  but 
instead  of  being  poured  out  it  is  rolled  in  a  nearly 
horizontal  position  under  a  cold  tap  or  on  a  block 
of  ice,  as  devised  by  Booker,  till  it  solidifies  as  a 
uniformly  thin  layer  on  the  inside  of  the  tube. 
Practically  we  deal  with  a  cylindrical  plate  of  gelatin  instead  of 
a  flat  one.  A  convenient  form  of  tube  for  this  method  is  one 


FlG.  21.  —  Koch's  levelling  apparatus, 
Hands  shown  in  second  position  just  as  th( 
plate  is  lowered  on  to  the  ground-glass  sur 
face.  By  executing  the  transference  of  tht 
plate  from  the  box  in  this  way,  the  surfact 


most  in  the  leveller,  and  thus  never  meets 
current  of  air  which  might  contaminate  it. 


FIG.  22.  — 
Esm  arch's 
tube  for  roll- 
culture. 


SEPARATION    OF   BACTERIA    BY  AGAR   MEDIA.  57 

with  a  constriction  a  short  distance  below  the  plug  of  cotton 
wool  (Fig.  22).  The  great  disadvantage  of  the  method  is,  that 
if  organisms  liquefying  the  gelatin  be  present,  the  liquefied 
gelatin  contaminates  the  rest  of  the  gelatin. 

Separation  by  Agar  Media.  —  i.  Agar  Plates.  —  The  only 
difference  between  the  technique  here  and  that  with  gelatin 
depends  on  the  difference  in  the  melting-points  of  the  two 
media.  Agar,  we  have  said,  melts  at  98°  C,  and  becomes  again 
solid  a  little  under  40°  C.  As  it  is  dangerous  to  expose  organ- 
isms to  a  temperature  above  42°  C.,  it  is  necessary  in  preparing 
tubes  of  agar  to  be  used  in  plate-cultures  to  first  melt  the  agar, 
by  boiling  in  a  vessel  of  water  for  a  few  minutes,  and  then  to 
cool  them  to  about  42°  C.  before  inoculating.  The  manipulation 
must  be  rapidly  carried  out,  as  the  margin  or  time,  before  solidi- 
fication occurs,  is  narrow  ;  otherwise  the  details  are  the  same  as 
for  gelatin.  Esmarch's  tubes  are  not  suitable  for  use  here,  as  the 
agar  does  not  adhere  well  to  the  sides.  If  to  the  agar  2  per 
cent  of  a  strong  watery  solution  of  pure  gum  arabic  is  added, 
Esmarch's  tubes  may,  however,  be  used. 

2.  Separation  by  Stroking  Mixture  on  Surface  of  Agar  Media. 
—  The  bacterial  mixture,  instead  of  being  mixed  in  the  medium, 
is  spread  out  on  its  surface.  The  method  may  be  used  both 
when  the  bacteria  to  be  separated  are  in  a  fluid,  and  when 
contained  in  a  fairly  solid  tissue  or  substance,  such  as  a  piece  of 
diphtheritic  membrane.  In  the  case  of  a  tissue,  for  example, 
a  small  proportion  entangled  in  the  loop  of  a  platinum  needle  is 
stroked  in  successive  parallel  longitudinal  strokes  on  sloped 
agar,  the  same  aspect  being  brought  in  contact  with  the  agar  in 
all  the  strokes.  Three  strokes  may  be  made  on  each  tube,  and 
three  tubes  are  usually  sufficient.  In  this  process  the  organisms 
on  the  surface  of  the  tissue  are  gradually  rubbed  off,  and  when 
growth  has  taken  place  it  will  be  found  that  in  the  later  strokes 
the  colonies  are  less  numerous  than  in  the  earlier,  and  sufficiently 
far  apart  to  enable  parts  of  them  to  be  picked  off  without  the 
needle  touching  any  but  one  colony.  When,  as  in  the  case  of 
diphtheritic  membrane,  putrefactive  organisms  are  likely  to  be 
present  on  the  surface  of  the  tissue,  these  can  be  in  great  part 
removed  by  washing  it  well  in  cold  water  previously  sterilised 
(vide  "  Diphtheria  ").  In  the  case  of  liquids,  the  loop  is  charged 
and  similarly  stroked.  Tubes  thus  inoculated  must  be  put  in 


58  METHODS    OF   CULTIVATION    OF    BACTERIA. 

the  incubator  in  the  upright  position  and  must  be  handled  care- 
fully so  that  the  condensation  water,  which  always  is  present  in 
incubated  agar  tubes,  may  not  run  over  the  surface.  Agar, 
poured  out  in  a  Petri's  dish  and  allowed  to  stand  till  firm,  may  be 
used  instead  of  successive  tubes.  Here  a  sufficient  number  of 
strokes  can  be  made  in  one  dish.  Sloped  blood-serum  tubes  may 
be  used  instead  of  agar.  The  method  is  rapid  and  easy  and 
gives  good  results. 

Separation  of  Pathogenic  Bacteria  by  Inoculation  of  Ani- 
mals. —  It  is  found  difficult  and  often  impossible  to  separate  by 
ordinary  plate  methods  certain  pathogenic  organisms,  such  as 
B.  tuberculosis,  B.  mallei,  and  the  pneumococcus,  when  such 
occur  in  conjunction  with  other  bacteria.  These  grow  best  on 
special  media,  and  the  first  two  (especially  the  tubercle  bacillus) 
grow  so  slowly  that  the  other  organisms  present  outgrow  them, 
cover  the  whole  plates,  and  make  separation  impossible.  The 
method  adopted  in  such  cases  is  to  inoculate  an  animal  with  the 
mixture  of  bacilli,  wait  until  the  particular  disease  develops,  kill 
the  animal,  and  with  all  aseptic  precautions  (vide  p.  120)  inocu- 
late tubes  of  suitable  media  from  characteristic  lesions  situated 
away  from  the  seat  of  inoculation,  e.g.  from  spleen  in  the  case 
of  B.  tuberculosis,  spleen  or  liver  in  the  case  of  B.  mallei,  and 
heart  blood  in  the  case  of  pneumococcus. 

Separation  by  killing  Non-spored  Forms  by  Heat. — This  is 
a  method  which  has  a  limited  application.  As  has  been  said, 
the  spores  of  a  bacterium  resist  heat  more  than  the  vegetative 
forms.  When  a  mixture  contains  spores  of  one  bacterium  and 
vegetative  forms  of  this  and  other  bacteria,  then  if  the  mixture 
be  boiled  for  a  few  minutes  all  the  vegetative  forms  will  be 
killed,  while  the  spores  will  remain  alive  and  will  develop  sub- 
sequently. This  method  can  be  easily  tested  in  the  case  of 
cultivating  B.  subtilis  from  hay  infusion.  A  little  chopped-up 
hay  is  placed  in  a  flask  of  water,  which  is  boiled  for  about  ten 
minutes.  On  this  being  allowed  to  cool  and  stand,  in  a  day  or 
two  a  scum  forms  on  the  surface,  which  is  found  to  be  a  pure 
culture  of  the  bacillus  subtilis.  The  method  is  also  often  used 
to  aid  in  the  separation  of  B.  tetani,  vide  infra. 


SEPARATION  OF  ANAEROBES. 


59 


THE  PRINCIPLES  OF  THE  CULTURE  OF  ANAEROBIC  ORGANISMS. 

All  ordinary  media,  after  preparation,  may  contain  traces  of 
free  oxygen,  and  will  absorb  more  from  the  air  on  standing. 
For  the  growth  of  anaerobes  this  oxygen  may  be  gotten  rid  of 
in  two  ways,  (i)  By  the  prolonged  passing  of  an  inert  gas, 
such  as  hydrogen,  through  the  medium  (liquefied  if  necessary), 
and  further,  the  medium  must  be  kept  in  an  atmosphere  of  the 
same  gas,  while  growth  is  going  on.  (2)  By  absorption  through 
the  use  of  pyrogallic  acid  in  an  alkaline  solution  as  described  by 
Buchner.  Media  for  anaerobes  may  be  kept  in  contact  with 
the  air,  if  they  contain  a  reducing  agent  which  does  not  inter- 
fere with  bacterial  growth.  Such  an  agent  takes  up  any  oxy- 
gen which  may  already  be  in  the  medium,  and  prevents  further 
absorption.  The  reducing  body  used  is  generally  glucose,  though 
formate  of  sodium  may  be  similarly  employed.  The  preparation 
of  such  media  has  already  been  described  (pp.  38,  40).  In  this 
case  the  medium  ought  to  be  of  considerable  thickness. 

The  Supply  of  Hydrogen  for  Anaerobic  Cultures.  — The  gas  is 
generated  in  a  large  Kipp's  apparatus  from  pure  sulphuric  acid 
and  pure  zinc. 
It  is  passed 
through  three 
wash-bottles,1 
as  in  Fig.  23. 
In  the  first  is 
placed  a  solu- 
tion of  lead  ace- 
tate (i  in  10 
of  water)  to 
remove  any 
traces  of  sul- 
phuretted hy- 
drogen. In  the 
second  is 
placed  a  i  in 

10  solution  of  silver  nitrate  to  remove  any  arsenietted  hydrogen 
which  may  be  present  if  the  zinc  is  not  quite  pure.  In  the 
third  is  a  10  per  cent  solution  of  pyrogallic  acid  in  caustic  potash 
solution  (i  :  10)  to  remove  any  traces  of  oxygen.  The  tube  lead- 


FlG.  23.  —  Apparatus  for  supplying  hydrogen  for  anaerobic  cultures. 

a.  Kipp's  apparatus  for  manufacture  of  hydrogen.  b.  Wash-bottle 
containing  i-io  solution  of  lead  acetate,  c.  Wash-bottle  containing  i-io 
solution  of  silver  nitrate  d.  Wash-bottle  containing  i-ro  solution  of 
pyrogallic  acid,  (b,  c,  and  d  are  intentionally  drawn  to  a  larger  scale  than 
a  to  show  details.) 


6o 


METHODS    OF   CULTIVATION    OF   BACTERIA. 


FlG.  24.  —  Esmarch's 
roll-tube  adapted  for  cul- 
ture containing  anaerobes. 


ing  from  the  last  bottle  to  the  vessel  containing  the  medium 
ought  to  be  sterilised  by  passing  through  a  Bunsen  flame,  and 
should  have  a  small  plug  of  cotton  wool  in  it  to  filter  the  hydro- 
gen germ-free. 

Separation  of  Anaerobic  Organisms.  —  (a)  By  Roll-tubes.  —  A 
i  \  inch  test-tube  has  as  much  gelatin  put  into  it  as  would  be 
,  i  used  in  the  Esmarch  roll-tube  method.     It 

is  corked  with  an  india-rubber  stopper  hav- 
ing two  tubes  passing  through  it,  as  in 
Fig.  24.  The  ends  of  the  tubes  are  partly 
drawn  out  as  shown,  and  covered  with  plugs 
of  cotton  wool.  Three  such  test-tubes  are 
prepared,  and  they  are  sterilised  in  the 
steam  steriliser  (p.  31).  After  sterilisation 
the  gelatin  is  melted  and  one  tube  inocu- 
lated with  the  mixture  containing  the 
anaerobes  ;  the  second  is  inoculated  from 
the  first,  and  the  third  from  the  second, 
as  in  making  ordinary  gelatin  plates. 
After  inoculation  the  gelatin  is  kept  liquid  by  the  lower  ends  of 
the  tubes  being  placed  in  water  at  about  30°  C.,  and  hydrogen  is 
passed  in  through  tube  x  for  twenty  minutes.  The  gas-supply 
tubes  are  then  completely  sealed  off  at  x  and  z,  and  each  test- 
tube  is  rolled  as  in  Esmarch's  method  till  the  gelatin  solidifies 
as  a  thin  layer  on  the  internal  surface.  A  little  hard  paraffin 
may  be  run  between  the  rim  of  the  test-tube  and  the  stopper, 
and  round  the  perforations  for  the  gas-supply  tubes,  to  ensure 
that  the  apparatus  is  air-tight.  The  gelatin  is  thus  in  an  atmos- 
phere of  hydrogen  in  which  the  colonies  may  develop.  The 
latter  may  be  examined  and  isolated  in  a  way  which  will  be 
presently  described.  The  method  is  admirably  suited  for  all 
anaerobes  which  grow  at  the  ordinary  temperature. 

(b)-  By  Novys  and  Buchner  s  Apparatus.  —  The  separation  of 
anaerobes  may  be  more  readily  carried  out  than  in  the  fore- 
going methods  by  making  dilutions  of  the  bacteria  in  agar, 
glucose  agar,  or  gelatin,  and  pouring  into  Petri's  dishes  and 
placing  them  in  either  a  Novy's  anaerobic  jar  (see  Fig.  25) 
or  in  a  modified  Buchner  apparatus.  If  one  uses  the  Novy 
jar,  the  air  is  displaced  by  allowing  a  good  stream  of  hydrogen 
gas  from  a  Kipp  generator  to  pass  through  it  for  at  least  30 


METHODS    OF   ANAEROBIC   CULTIVATION. 


61 


minutes  before  closing  the  cock  in  the  top  of  the  jar.  In  effect- 
ing displacement  of  the  air  it  is  necessary  to  observe  to  adjust 
the  cock  so  that  the  gas  enters  directly 
into  the  jar  from  above  and  finds  its 
way  out  through  the  combined  rub- 
ber and  glass  tubing  adjustment  from 
below. 

In  the  alternative  method,  use  is 
made  of  an  ordinary  chemical  desic- 
cating jar  of  suitable  size,  6  inches  in 
diameter,  in  the  lower  compartment  of 
which  is  placed  about  150  c.c.  of  a  I 
per  cent  sodium  hydrate  solution  in 

Which      is      dissolved      IO     grammes     Of      FlG- 2S--Novys  anaerobic  jar. 

pyrogallic  acid.  Into  the  upper  portion  of  the  jar  are  placed 
the  Petri's  dish  cultures,  and  at  once  the  cover  of  the  vessel,  pre- 
viously smeared  with  vaseline  on  its  contact  surface,  is  firmly 
affixed.  These  vessels  are  then  placed  for  forty-eight  hours  in 
the  thermostat  before  examining. 

(c)  By  Bullock's  Apparatus  for  Anaerobic  Culture.  —  This  can 
be  recommended  for  plating^ut  mixtures  containing  anaerobes, 

and  for  obtaining  growths  (especially 
surface  growths)  of  the  latter.  It 
consists  (Fig.  26)  of  a  glass  plate  as 
base  on  which  a  bell-jar  can  be  firmly 
luted  down  with  unguentum  resinae. 
In  the  upper  part  of  the  bell-jar  are 
two  apertures  furnished  with  ground 
stoppers,  and  through  each  of  the  lat- 
ter passes  a  glass  tube  on  which  is  a 
stopcock.  One  tube,  bent  slightly 
just  after  passing  through  the  stopper, 
extends  nearly  to  the  bottom  of  the 
chamber ;  the  other  terminates  imme- 
diately below  the  stopper.  In  using 
the  apparatus  there  is  set  on  the  base- 
plate a  shallow  dish,  of  slightly  less  diameter  than  that  of  the  bell- 
jar,  and  having  a  little  heap  of  from  two  to  four  grammes  of  dry 
pyrogallic  acid  placed  in  it  towards  one  side.  Culture  plates 
made  in  the  usual  way  can  be  stacked  on  a  frame  of  glass  rods 


FIG.  26.  —  Bulloch's  apparatus  for 
anaerobic  plate-cultures. 


62  METHODS    OF   CULTIVATION   OF   BACTERIA. 

resting  on  the  edges  of  the  dish,  or  a  beaker  containing  culture 
tubes  can  be  placed  in  it.  The  bell-jar  is  then  placed  in  posi- 
tion so  that  the  longer  glass  tube  is  situated  over  that  part  of 
the  bottom  of  the  shallow  dish  farthest  away  from  the  pyrogallic 
acid,  and  the  bottom  and  stoppers  are  luted.  The  air  in  the  bell- 
jar  is  now  expelled  by  passing  a  current  of  coal-gas  through 
the  short  glass  tube,  and  both  stoppers  are  closed.  A  partial 
vacuum  is  then  effected  in  the  jar  by  connecting  the  short  tube 
up  with  an  air-pump,  opening  the  tap,  and  giving  a  few  strokes 
of  the  latter.  A  solution  of  7  grammes  solid  caustic  potash  dis- 
solved in  145  c.c.  water  is  made,  and  into  the  vessel  containing 
it  a  rubber  tube  connected  with  the  long  glass  tube  is  made  to 
dip,  and  the  stopper  of  the  latter  being  opened,  the  fluid  is  forced 
into  the  chamber,  spreads  over  the  bottom  of  the  shallow  dish, 
and,  coming  in  contact  with  the  acid,  sodium  pyrogallate  is 
formed  which  absorbs  any  free  oxygen  still  present.  Before  the 
whole  of  the  fluid  is  forced  in,  the  rubber  tube  is  placed  in  a 
little  water,  and  this,  passing  through  the  glass  tubes,  washes 
out  the  soda  and  prevents  erosion  of  the  glass.  The  whole 
apparatus  may  be  placed  in  the  incubator  till  growth  occurs. 

It  is  often  advisable  in  dealing  with  material  suspected  to 
contain  anaerobes  to  inoculate  an  ordinary  deep  glucose  agar 
tube  with  it,  and  incubating  for  24  or  48  hours,  to  then  apply  an 
anaerobic  separation  method  to  the  resultant  growth.  Some- 
times the  high  powers  of  resistance  of  spores  to  heat  may  be 
taken  advantage  of  in  aiding  the  separation  (vide  "  Tetanus  "). 

Cultures  of  Anaerobes.  —  When  by  one  or  other  of  the  above 
methods  separate  colonies  have  been  obtained,  growth  may  be 
maintained  on  media  in  contact  with  ordinary  air.  The  media 
generally  used  are  those  which  contain  reducing  agents,  and  the 
test-tubes  containing  the  medium  must  be  filled  to  a  depth  of  4 
inches.  They  are  sterilised  as  usual  and  are  called  "deep" 
tubes.  The  long  straight  platinum  wire  is  used  for  inoculating, 
and  it  is  plunged  well  down  into  the  "  deep  "  tube.  A  little  air 
gets  into  the  upper  part  of  the  needle  track,  and  no  growth 
takes  place  there,  but  in  the  lower  part  of  the  needle  track 
growth  occurs.  From  such  "deep"  cultures  growths  may  be 
maintained  indefinitely  by  successive  sub-cultures  in  similar 
tubes.  Even  ordinary  gelatin  and  agar  can  be  used  in  the  same 
way  if  the  medium  is  heated  to  boiling-point  before  use  to  expel 
any  absorbed  oxygen. 


ANAEROBIC   CULTURE    IN    LIQUID    MEDIA. 


Cultures  of  Anaerobes  in  Liquid  Media.  —  It  is  necessary  to 
employ  such  in  order  to  obtain  the  toxic  products  of  the  growth 
of  anaerobes.  Glucose 
broth  is  most  convenient. 
It  is  placed  either  (i)  in  a 
conical  flask  with  a  lateral 
opening  and  a  perforated 
india-rubber  stopper, 
through  which  a  bent  glass 
tube  passes,  as  in  Fig.  27,  a, 
by  which  hydrogen  may  be 
delivered,  or  (2)  in  a  conical 
flask  with  a  rubber  stopper  a 

furnished  with  two  holes,  as  ,?,,,, 

a.    rlask   lor  anaerobes   in    liquid   media.      Lateral 

in  Fig.  27,  b,  thrOUgh  a  tube    nozzle  and  stopper  fitted  for  hydrogen  supply,    b.  A  stop- 
r  .       per  arranged  for  a  flask  without  lateral  nozzle. 

in  one  of  which  hydrogen  is 

delivered,  while  through  the  tube  in  the  other  the  gas  escapes. 
The  inner  end  of  the  gas  delivery  tube  must  in  either  case  be 
below  the  surface  of  the  liquid  ;  the  inner  end  of  the  lateral 
nozzle  in  the  one  case,  "and  the  inner  end  of  the  escape  tube  in 
the  other,  must  of  course  be  above  the  surface  in  the  liquid. 
The  single  tube  in  the  one  case  and  the  two  tubes  in  the  other 
ought  to  be  partially  drawn  out  in  a  flame  to  facilitate  subsequent 
complete  sealing.  The  ends  of  the  tubes  through  which  the  gas 
is  to  pass  are  previously  protected  by  pieces  of  cotton  wool  tied 
on  them.  It  is  well  previously  to  place  in  the  tube,  through 
which  the  hydrogen  is  to  be  delivered,  a  little  plug  of  cotton 
wool.  The  flask  being  thus  prepared,  it  is  sterilised  by  methods 
B  (2)  or  B  (3).  On  cooling  it  is  ready  for  inoculation.  In  the 
case  of  the  flask  with  the  lateral  nozzle,  the  cotton-wool  covering 
having  been  momentarily  removed  a  wire  charged  with  the 
organism  is  passed  down  to  the  bouillon.  In  the  other  kind  of 
flask  the  stopper  must  be  removed  for  an  instant  to  admit  the 
wire.  The  flask  is  then  connected  with  the  hydrogen  apparatus 
by  means  of  a  short  piece  of  sterile  india-rubber  tubing,  and 
hydrogen  is  passed  through  for  half  an  hour.  In  the  case  of 
flask  (i),  the  lateral  nozzle  is  plugged  with  molten  paraffin 
covered  with  alternate  layers  of  cotton  wool  and  paraffin,  the 
whole  being  tightly  bound  on  with  string.  The  entrance  tube 
is  now  completely  drawn  off  in  the  flame  before  being  discon- 


64 


METHODS   OF   CULTIVATION    OF    BACTERIA. 


nected  from  the  hydrogen  apparatus.  In  the  case  of  flask  (2), 
first  the  exit  tube  and  then  the  entrance  tube  are  sealed  off  in  the 
flame  before  the  flask  is  disconnected  from  the  hydrogen  ap- 
paratus. It  is  well  in  the  case  of  both  flasks  to  run  some  melted 
paraffin  all  over  the  rubber  stopper.  Sometimes  much  gas  is 
evolved  by  anaerobes,  and  in  dealing  with  an  organism  where 
this  will  occur,  provision  must  be  made  for  its  escape.  This  is 
conveniently  done  by  leading  down  the  exit  tube,  and  letting 
the  end  just  dip  into  a  trough  of  mercury  (Fig.  28),  or  into  mer- 
tt  cury  in  a  little  bottle  tied  on  to  the 

end  of  the  exit  tube.  The  press- 
ure of  gas  within  causes  an  escape 
at  the  mercury  contact,  which  at 
the  same  time  acts  as  an  efficient 
valve.  The  method  of  culture  in 
fluid  media  is  used  to  obtain  the 
soluble  products  of  such  anaerobes 
as  the  tetanus  bacillus. 

Dr.  W.  H.  Park  of  New  York 
has  recently  introduced  a  very 
simple  method  for  making  anae- 

TQ^C       cultures        in        fluid       media. 

An  Erlenmeyer  flask  containing 
a  suitable  quantity  of  medium  is  boiled  in  a  water  bath  for 
ten  or  fifteen  minutes,  to  drive  off  all  dissolved  oxygen,  then 
rapidly  cooled  down  and  inoculated.  Hot  melted  paraffin  is 
now  poured  into  the  flask  until  it  reaches  a  depth  of  2  or  3  mm., 
and  upon  solidifying  it  forms  a  perfect  seal,  excluding  the  air 
completely,  yet  not  adhering  to  the  glass  so  strongly  as  to 
prevent  escape  of  gases  should  any  be  formed  by  the  growing 
anaerobes. 

Wright's  Method  of  Anaerobic  Culture.  —  Utilising  Buchner's 
pyrogallic  acid  medium  for  the  removal  of  oxygen,  Wright  has 
contrived  two  very  ingenious  methods  for  anaerobic  culture  in 
ordinary  test-tubes.  In  the  first  method,  which  is  applicable  to 
both  solid  and  fluid  media  (Fig.  29),  after  inoculating,  the  cotton 
plug,  always  made  of  absorbent  cotton,  is  cut  off  flush  with  the 
extremity  of  the  tube,  and  pushed  inwards  to  a  distance  of  i  cm. 
It  is  then  impregnated  with  i  c.c.  of  a  watery  solution  of  pyro- 
gallic acid  (freshly  prepared  by  adding  to  one  part  of  water  an 


FIG.  2s.-  Fiask  arranged  for  culture  of 
anaerobes  which  develop  gas. 

b   is   a  trough  of  mercury  into  which   exit 
tube  dips. 


WRIGHT'S    METHOD    OF    ANAEROBIC   CULTURE. 


approximately  equal  bulk  of  pyrogallic  acid),  and  adding  to  that 

i  c.c.  of  a  5  percent  solution  of  sodium  hydrate.    A  rubber  stopper 

is  next  tightly  fitted  into  the  end  of  the 

tube,  and  the  apparatus  is  then  ready 

for   incubating.      The   second  method, 

suitable  for  fluid  media  only,  consists  of 

a  system  of  soft  rubber  and  glass  tubes 

arranged  in  an  ordinary  culture  tube,  as 

shown  in  Fig.  30.     The  small  glass  tube 

A  is  drawn  out  slightly  at  both  ends 

and  capped  by 
pieces  of  soft 
rubber  tubing, 
into  one  of 
which  is  in- 
serted a  short 
length  of  glass 
tubing  of  a 
much  smaller 
calibre  than  A, 
and  which  is 
plugged  with 
cotton  at  its  up- 
per end,  where 
a  small  piece 

II  I  of   rubber   tub- 

Al  IHfill         ing    is    affixed. 

Into  the  tube 
containing 

such  an  apparatus  the  usual  quantity 
of  bouillon  is  poured,  and  the  whole  is 
sterilised  by  steam,  care  being  taken 
that  the  rubber  portions  are  not  bent. 
To  prepare  a  culture  it  is  advisable  to 
boil  the  broth  over  a  free  flame  before 
inoculating,  so  as  to  drive  off  as  much 
dissolved  oxygen  as  possible,  then  im- 
merse the  tube  in  cold  water.  When 

cool,  inoculate  the  bouillon  and  suck  it  up  into  the  system  of 

tubes  well  above  C,  then,  pinching  E  between  the  fingers  to 


By  permission,  from  Mallory  & 
Wright's  "  Pathological  Technique." 

FIG.  29.  — Wright's  method 
for  the  cultivation  of  anaerobes 
in  solid  media. 


By  permission,  from  Mallory  & 
Wright's  "  Pathological  Technique." 

FIGS.  30,  31.  —  Wright's 
method  for  the  cultivation  of 
anaerobes  in  flilid  media. 


66  METHODS    OF   CULTIVATION    OF   BACTERIA. 

prevent  back  flow  of  the  fluid,  press  downwards  so  as  to  buckle 
up  the  tubing  at  B  and  C  (Fig.  31).  This  forms  both  a  water 
and  air  tight  compartment  in  A,  and  readily  allows  anaerobic 
growth. 

When  it  is  desired  to  grow  anaerobes  on  the  surface  of  a 
solid  medium  such  as  agar,  tubes  of  the  form  shown  in  Fig.  32, 

a  and  b,  may  be 
used-  A  stroke 
culture  having 
been  made,  the 
air  is  replaced  by 
hydrogen  as  just 

FIG.  32.  —  Tubes  for  anaerobic  cultures  on  the  surface  of  solid      described,        and 

media-  the     tubes     are 

fused  at  the  constrictions.  Such  a  method  is  of  great  value 
when  it  is  required  to  get  the  bacteria  free  from  admixture  of 
medium,  as  in  the  case  of  staining  flagella.  By  the  use  of 
Novy's  jar  anaerobes  may  be  made  to  grow  on  all  media  in 
ordinary  culture  tubes. 

MISCELLANEOUS  METHODS. 

Collodion,  or  Celloidin  Sacs.  —  These  were  introduced  by 
Metschnikoff,  Roux,  and  Salimbeni  during  their  studies  on  the 
spirillum  of  Asiatic  cholera,  and  further  exploited  by  Nocard 
and  Roux  in  their  research  upon  the  cause  of  pleuro-pneumonia 
of  cattle.  The  usefulness  of  such  a  sac  lies  in  the  fact  that  liv- 
ing cultures  can  be  introduced  into  the  body  of  an  animal  with- 
out coming  into  direct  contact  with  its  tissues  or  body  fluids, 
whilst  their  soluble  products  can,  through  the  osmotic  proper- 
ties of  the  collodion,  pass  outwards  and  be  absorbed  by  the  tis- 
sues, and  at  the  same  time  the  animal  fluids  can  pass  within  and 
nourish  the  bacteria.  Thus  a  ready  means  is  afforded  of  exalt- 
ing virulence  of  bacteria,  of  producing  agglutinative  and  other 
phenomena,  without  great  difficulties  being  thrown  in  the  way, 
such  as  otherwise  might  happen.  The  method  used  by  the  French 
investigators  in  making  the  sacs,  as  stated  by  Novy,  is  rather 
an  involved  and  laborious  proceeding,  and  lately  a  newer  and 
easier  method  has  been  recommended  by  McCrae,  wherein  gela- 
tin capsules  are  coate^l  with  collodion  and  the  resulting  sac  is 
mounted  on  glass  tubing  and  prepared  for  final  use.  McCrae's 


METHOD   OF   MAKING  COLLODION   SACS.  6/ 

method  has  been  further  improved  upon  by  Harris,  whose 
scheme  is  briefly  given  as  follows :  — 

To  the  smaller  end  of  a  gelatin  capsule  such  as  is  used  by 
veterinary  surgeons  (Fig.  33,  A),  a  piece  of  glass  tubing  about 
4  cm.  long  and  3  mm.  in  inside  diameter  is  affixed  by  gently 
heating  the  tubing  and  causing  it  to  adhere  (Fig.  33,  B).  The 
bore  of  the  tubing  is  then  cleared  of  obstructing  gelatin,  the 
junction  of  glass  and  gelatin  is  painted  around  with  a  solution 
of  moderately  thick  collodion  or  celloidin  and  allowed  to  dry. 
Then  the  whole  capsule  is  dipped  in  the  solution,  removed,  and 
rotated  so  as  to  give  a  perfectly  even  coating  to  the  capsule. 
When  set,  the  capsule  is  allowed  to  thoroughly  dry,  and  if 
necessary  is  again  coated  and 
dried,  and  is  reinforced  at 
three  points  (Fig.  33,  C)  and 
once  more  dried.  The  gelatin 
is  removed  by  filling  up  the 
capsule  with  water  by  means 
of  a  fine  bore  pipette  of  the 
Pasteur  type,  and  boiled  a 
couple  of  minutes  in  a  'pan 
of  water,  then  the  gelatin  is 

A.   Empty  gelatin  capsule,  actual  size.     "  No.  12 
SUCked    OUt     by    means    Of    the       Veterinary,"  P.  D.  &  Co. 

•1,1  •  .cni  B.    Glass  tube  sealed  into  end  of  capsule. 

pipette  and  the  sac  is  refilled       c>  Sac  ready  for  insertion.  a>  ^  points  of 

With  bouillon.        Sterilisation  is       reinforcement  ;  d,  limit  for  height  of  the  column 

of  fluid  inside. 

effected  by  placing  the  sac  in 

a  tube  of  broth,  sac  end  uppermost,  with  enough  broth  in  it 
to  cover  the  sac  to  the  depth  of  I  cm.,  and  either  autoclaving 
for  five  minutes  under  one  atmosphere  pressure  or  by  steaming 
for  three  successive  days  in  the  Arnold  steriliser.  The  sac  is 
inoculated  by  first  removing  some  of  its  contents  (Fig.  33  C) 
under  aseptic  conditions,  and  then  introducing  a  small  quantity 
of  the  desired  bacterium  in  suspension,  or  in  fluid  culture,  by 
means  of  a  sterile  pipette ;  it  is  then  to  be  seized  with  sterilised 
forceps  by  the  glass  shank,  and  the  latter  brought  in  contact 
with  the  small  flame  of  a  blast  lamp  and  sealed  off  (see  Fig.  33, 
C).  The  inoculated  sac  is  next  to  be  tested  for  tightness 
by  incubating  it  in  a  bouillon  culture  tube,  after  first  washing 
it  thoroughly  with  sterile  water  to  remove  any  organisms  which 
may  have  alighted  upon  it  during  the  exposure  to  the  air 


B 

FIG.  33. 


68 


METHODS    OF   CULTIVATION    OF   BACTERIA. 


consequent  upon  inoculating  and  sealing  procedures ;  if  after 
incubation  no  growth  occurs  in  the  broth  outside  the  capsule, 
it  is  then  ready  for  insertion  into  the  abdominal  cavity  of  the 
animal. 

Hanging-drop  Cultures.  —  It  is  often  necessary  to  observe 
micro-organisms  alive,  either  to  watch  the  method  and  rate  of 
their  multiplication,  or  to  investigate  whether  or  not  they  are 
motile.  This  is  effected  by  making  hanging-drop  cultures.  The 
method  in  the  form  to  be  described  is  only  suitable  for  aerobes. 
For  this  special  slides  are  necessary.  Two  forms  are  in  use  and 
are  shown  in  Fig.  34.  In  A  there  is  ground  out  on  one  surface 


2> 


FIG.  34. 

A,  A.    Hollow-ground  slide  for  hanging-drop  cultures  shown  in  plan  and  section. 
B.    Another  form  of  slide  for  similar  cultures. 

a  hollow  having  a  diameter  of  about  half  an  inch.  That  shown 
in  B  explains  itself.  The  slide  to  be  used  and  a  cover-glass  are 
sterilised  by  hot  air  in  a  Petri's  dish,  or  simply  by  being  heated 
in  a  Bunsen  and  laid  in  a  sterile  Petri  to  cool.  In  the  case  of 
A  one  or  other  of  two  manipulation  methods  may  be  employed, 
(i)  If  the  organism  be  growing  in  a  liquid  culture,  a  loop  of  the 
liquid  is  placed  on  the  middle  of  the  under  surface  of  the  sterile 
cover-glass,  which  is  held  in  forceps,  the  points  of  which  have 
been  sterilised  in  a  Bunsen  flame.  If  the  organism  be  growing 
in  a  solid  medium,  a  loopful  of  sterile  bouillon  is  placed  on  the 
cover-glass  in  the  same  position,  and  a  very  small  quantity  of 
the  culture  (picked  up  with  a  platinum  needle)  is  rubbed  up  in 
the  bouillon.  The  edge  of  the  hollow  is  smeared  with  vaseline, 
or  immersion  oil,  the  cover  is  then  carefully  lowered  over  the 


HANGING-DROP   PREPARATIONS  69 

cell  on  the  slide,  the  drop  not  being  allowed  to  touch  the  wall  or 
the  edge  of  the  cell,  and  the  preparation  is  then  complete  and  may 
be  placed  under  the  microscope.  If  necessary,  it  may  be  first 
incubated  and  then  examined  on  a  warm  stage.  (2)  The  sterile 
cover-glass  is  placed  on  a  sterile  plate  (an  ordinary  glass  plate 
used  for  plate-cultures  is  convenient).  The  drop  is  then  placed 
on  its  upper  surface,  the  details  being  the  same  as  in  the  last 
case.  The  edge  of  the  cell  in  the  slide  is  then  painted  with 
vaseline,  and  the  slide,  held  with  the  hollow  surface  downwards, 
is  lowered  on  to  the  cover-glass,  to  the  rim  of  which  it  of  course 
adheres.  The  slide  with  the  cover  attached  is  then  quickly 
turned  right  side  up,  and  the  preparation  is  complete. 

In  the  case  of  B  the  drop  of  fluid  is  placed  on  the  centre  of 
the  table  x.  The  drop  must  be  thick  enough  to  come  in  contact 
with  the  cover-glass  when  the  latter  is  lowered  on  the  slide,  and 
not  large  enough  to  run  over  into  the  surrounding  trench  y. 
The  cover-glass  is  then  lowered  on  to  the  drop,  and  vaseline  is 
painted  along  the  margin  of  the  cover-glass.  The  method  of 
microscopic  examination  is  described  on  page  87. 

Anaerobic  Hanging-drop  Cultures.  —  The  growth  and  exami- 
nation of  bacteria  in  hanging-drops  under  anaerobic  conditions 
involve  considerable  difficulty,  but  may  be  carried  out  in 
an  apparatus  devised  by  A 

Graham  Brown  (Fig.  35). 
It  consists  of  two  brass 
plates  (a  and  a')  which 
can  be  approximated  by 
screws,  and  which  have  two 
rounded  apertures  in  their  c 
middle,  f  inch  in  diameter. 

:  / 

These  support  two  rubber  f  d  e 

rings,     an     upper     thinner      FIG.  35.  — Graham  Brown's  chamber  for  anaerobic 

one  (b)  and  a  lower  thick  hanging-drops. 

,    .        ,      .      .  ,.  (A  portion  of  one  edge  of  upper  plate  is  shown  cut  away.) 

one  (a),  their  inner  diame- 
ter being  the  same  as  that  of  the  apertures  in  the  plates.  Between 
b  and  d  is  placed  a  stout  cover-glass  of  suitable  size  (c) ;  d  is 
separated  from  the  plate  a1  by  a  square  plate  of  glass  e  (a  por- 
tion of  an  ordinary  glass  slide  for  microscopical  purposes  does 
well).  Two  small  metal  tubes  /  are  inserted  through  the  rub- 
ber d.  Method  of  use :  Fix  up  the  apparatus  as  shown  above, 


70  METHODS   OF   CULTIVATION   OF   BACTERIA. 

the  screws  being  just  tight  enough  to  keep  the  parts  in  position, 
and  sterilise  in  the  steam  steriliser.  Screw  up  more  firmly  so 
as  to  make  the  rubber  bulge  slightly.  Fill  a  hypodermic  syringe 
with  some  sterile  glucose  bouillon,  push  the  needle  through  the 
rubber  d,  and,  tilting  the  point  of  the  needle  against  the  glass  c, 
slowly  inject  enough  to  form  a  drop  on  the  under  surface  of  c. 
Withdraw  the  syringe  and  inoculate  its  point  with  the  bacterium, 
again  introduce  and  inoculate  the  drop.  Pass  hydrogen  through 
one  of  the  tubes  for  fifteen  minutes,  close  the  ends  of  the  tubes, 
and  incubate  at  the  required  temperature.  The  apparatus  can 
be  put  on  the  stage  of  a  microscope  and  examined  from  time 
to  time. 

Hanging-block  Cultures.  —  Applying  the  principle  of  the 
hanging-drop  culture  to  solid  media,  Hill  has  introduced  a  new 
method  for  the  more  accurate  study  of  the  morphology  and 
development  of  bacteria,  which  is  equally  applicable  to  aerobic 
or  anaerobic  forms.  Ordinary  plain  agar  is  melted  down  and 
poured  into  a  Petri's  dish  to  the  depth  of  one-quarter  of  an  inch 
and  solidified  ;  a  piece  is  then  cut  out  of  it  about  one-quarter  or 
one-third  of  an  inch  square  and  affixed  to  a  sterile  slide.  The 
upper  surface  of  this  block  is  then  inoculated  with  a  bouillon 
culture  of  the  selected  organism  in  the  manner  of  making  a 
cover-slip  preparation,  and  the  whole  is  covered  with  a  small 
sterile  dish  and  set  in  the  thermostat  to  dry  for  five  or  ten 
minutes.  This  step  being  completed,  a  sterile  cover-slip  is 
placed  upon  the  inoculated  surface,  avoiding  as  far  as  possible 
the  imprisonment  of  air-bubbles,  and  cemented  down  with 
melted  agar  by  means  of  a  platinum  loop.  The  cover-slip  with 
the  agar  block  is  now  to  be  removed  from  the  slide  and  sealed 
to  a  moist  chamber  with  paraffin ;  the  preparation  can  now  be 
studied  at  room  temperature,  or  transferred  to  a  warm  stage. 
For  the  observation  of  anaerobes  an  alkaline  solution  of  pyro- 
gallol  may  be  introduced  into  the  moist  chamber,  which  is  then 
made  air-tight. 

Thermal  Death-point  Test.  —  It  is  sometimes  necessary  to 
determine  the  death-point  of  a  bacterium  by  exposure  to  moist 
heat.  This  is  most  accurately  performed  by  the  aid  of  Stern- 
berg's  glass  bulbs  made  in  the  fashion  shown  in  Fig.  36.  A 
twenty-four-hour-old  broth  culture  of  the  given  bacterium,  pre- 
pared beforehand,  is  to  be  poured  out  into  a  sterile  Petri's  dish, 


DETERMINATION    OF   THERMAL   DEATH-POINT.          71 

then  having  taken  a  bulb  and  sterilised  the  point  and  broken  it 
off  with  sterile  forceps,  the  bulbous  end  is  to  be  rapidly  passed 
through  the  flame  of  a  Bunsen  burner  four  or  five  times  to 
expel  some  of  the  air,  and  the  sterile  point  of  the  shank  is 
to  be  dipped  into  the  fluid  in 
the  dish,  and  as  the  bulb  cools 
the  fluid  runs  slowly  up  the 
shank  and  falls  into  the  bulb 

below.        It    is    well    not    tO    fill         FIG.  36.  —  Sternberg's  bulb  adapted  for 

the  bulb  more  than  one-quarter,  thermal  death-point  test. 

as  a  great  bulk  of  fluid  is  to  be  avoided,  interfering  as  it  does  with 
the  delicacy  of  the  test.  Removing  the  bulb  from  the  fluid,  its 
point  is  carefully  sealed  in  the  flame  and  it  is  then  deposited  in 
a  small  galvanised  sheet-iron  box  perforated  with  many  small 
holes,  or  into  a  stout,  finely  meshed  wire  box;  both  bulb  and 
box  are  then  to  be  placed  in  a  water  bath  with  enough  water 
in  it  to  submerge  the  box  to  the  depth  of  at  least  one  inch,  and 
kept  for  the  required  time  at  a  constant  temperature.  [In 
testing  vegetative  forms  of  bacteria,  it  is  recommended  to  begin 
with  an  exposure  of  five  minutes  at  50°  C.,  then  ten  minutes  at 
50°  C.,  and  so  on,  for  every  five  succeeding  degrees  up  to  65°. 
Spores  are  tested  in  boiling  water  with  exposures  varying  from 
one  minute  up  to  twenty,  or  more.]  After  conditions  of  time 
and  temperature  have  been  fulfilled,  the  bulb  is  removed,  the 
shank  wiped  dry,  the  point  broken  off  by  forceps  under  sterile 
precautions,  and  the  shank  grasped  by  the  forceps  near  the  bulb, 
which  is  now  held  uppermost  so  as  to  permit  of  the  ready  dis- 
charge of  the  contents.  This  step  is  accomplished  by  introduc- 
ing the  shank  of  the  bulb  into  a  tube  of  previously  melted  agar, 
whose  temperature  is  42°  C.,  and,  bringing  the  upper  empty  end 
of  the  bulb  near  to  the  lowermost  portion  of  Bunsen  flame, 
expansion  of  the  air  at  once  drives  the  contents  into  the  agar, 
when  they  are  to  be  well  mixed  and  poured  into  a  sterile  Petri's 
dish,  and  incubated  for  72  hours,  and  examined  for  evidences 
of  growth.  Caution  must  be  observed  in  expelling  the  contents 
of  the  bulb,  lest  the  flame  come  into  direct  contact  and  vitiate 
the  experiment. 

The  Counting  of  Colonies.  —  An  approximate  estimate  of  the 
number  of  bacteria  present  in  a  given  amount  of  a  fluid  (say, 
water)  can  be  arrived  at  by  counting  the  number  of  colonies 


72  METHODS   OF   CULTIVATION   OF   BACTERIA. 

which  develop  when  that  amount  is  added  to  a  tube  of  suitable 
medium,  and  the  latter  plated  and  incubated.  An  ordinary 

plate  should  be  used  in  such 
a  case,  and  the  medium  poured 
out  in  as  rectangular  a  shape 
as  possible.  For  the  counting, 
an  apparatus  such  as  is  shown 
in  Fig.  37  is  employed.  This 
consists  of  a  sheet  of  glass  ruled 
into  squares  as  indicated,  and 
supported  by  its  corners  on 

FIG.  37.  —  Apparatus  for  counting  colonies 

(Wolff hugei's).  wooden  blocks.     The  table  to 

which  these  blocks  are  attached 

has  a  dark  surface.  The  plate-culture  containing  the  colonies 
is  laid  on  the  top  of  the  ruled  glass.  The  number  of  colonies 
in,  say,  twenty  of  the  smaller  squares,  is  then  counted,  and 
an  average  struck.  The  total  number  of  squares  covered  by 
the  medium  is  then  taken,  and  by  a  simple  calculation  the 
total  number  of  colonies  present  can  be  obtained.  Plate-cul- 
tures in  Petri's  dishes  are  sometimes  employed  for  purposes  of 
counting.  The  bottoms  of  such  dishes  are,  however,  never 
flat,  and  the  thickness  of  the  medium  thus  varies  in  different 
parts.  If  these  dishes  are  to  be  used,  a  circle  of  the  same 
size  as  the  dish  can  be  drawn  with  Chinese  white  on  a  black 
card,  the  circumference  divided  into  equal  arcs,  and  radii  drawn. 
The  dish  is  then  laid  on  the  card,  the  number  of  colonies  in  a 
few  of  the  sectors  counted,  and  an  average  struck  as  before. 
In  counting  colonies  it  is  always  best  to  aid  the  eye  with  a  small 
hand-lens. 

The  Bacteriological  Examination  of  the  Blood. — (a)  This 
may  be  done  by  taking  a  small  drop  from  the  skin  surface,  e.g. 
the  lobe  of  the  ear.  The  part  should  be  thoroughly  washed 
with  i-iooo  corrosive  sublimate  and  dried  with  sterile  cotton 
wool.  It  is  then  washed  with  absolute  alcohol  to  remove  the 
antiseptic,  drying  being  allowed  to  take  place  by  evaporation. 
A  prick  is  then  made  with  a  sterile  surgical  needle  ;  the  drop 
of  blood  is  caught  with  a  sterile  platinum  loop  and  smeared  on 
the  surface  of  agar  or  blood  serum.  Film  preparations  for 
microscopic  examination  may  be  made  at  the  same  time.  It  is 
rare  to  obtain  growths  from  the  blood  of  the  human  subject 


METHOD    OF   PERFORMING  LUMBAR   PUNCTURE.          73 

under  such  conditions  (vide  special  chapters),  and  if  colonies 
appear  the  procedure  should  be  repeated  to  exclude  the  pos- 
sibility of  accidental  contamination. 

(b)  But  when  it  is  possible  a  much  greater  quantity  of  blood 
should  be  obtained  to  make  the  examination  of  any  real  value. 
A  vein  at  the  bend  of  the  elbow  affords  the  readiest  opportunity 
of  getting  blood.  One  proceeds  as  follows :  A  bandage  is 
applied  tightly  around  the  middle  third  of  the  upper  arm,  and 
the  skin  over  the  selected  area  is  rendered  as  aseptic  as  possible 
by  scrubbing  with  hot  water  and  soap,  washing  off  the  soap 
with  sterile  water,  wiping  with  alcohol  and  ether,  and  rubbing 
well  with  bichloride  of  mercury,  i-iooo.  Then  the  operator, 
having  disinfected  his  hands,  enters  a  swollen  vein  with  the 
point  of  a  syringe  of  10-15  c-c-  capacity  and  withdraws  not 
less  than  10  c.c.  of  blood.  The  puncture  made  by  the  syringe 
is  closed  with  sterile  absorbent  cotton  and  collodion.  The 
blood  must  be  quickly  distributed  amongst  six  tubes  of  melted 
agar  and  plated,  or  divided  between  five  flasks  containing 
150-200  c.c.  of  sterile  broth,  and  these  incubated  for  two  or 
three  days  and  then  subjected  to  study. 

In  examining  the  blood  of  the  spleen  a  portion  of  the  skin 
over  the  organ  is  sterilised  in  the  same  way,  a  few  drops  are 
withdrawn  from  the  organ  by  a  sterile  hypodermic  syringe  and 
cultures  made.  (For  microscopic  methods,  vide  p.  88  et  seq.) 

Bacteriological  Examination  of  the  Cerebro-spinal  Fluid — 
Lumbar  Puncture.  —  This  diagnostic  procedure,  which  is  some- 
times called  for  in  cases  of  meningitis,  can  be  carried  out  with 
a  sterilised  "  antitoxin  needle  "  as  follows  :  The  patient  should 
lie  on  the  right  side,  with  knees  somewhat  drawn  up  and  left 
shoulder  tilted  somewhat  forward,  so  that  the  back  is  fully 
exposed.  The  skin  over  the  lumbar  region  is  then  carefully 
sterilised,  as  above  described,  and  the  hands  of  the  operator 
should  be  similarly  treated.  The  spines  of  the  lumbar  vertebrae 
having  been  counted,  the  left  thumb  or  forefinger  is  pressed  into 
the  space  between  the  3rd  and  4th  spines  in  the  middle  line  ;  the 
needle  is  then  inserted  about  half  an  inch  to  the  right  of  the 
middle  line  at  this  level  and  pushed  through  the  tissues,  its 
course  being  directed  slightly  inwards  and  upwards,  till  it  enters 
the  sub-arachnoid  space.  When  this  occurs  fluid  passes  along 
the  needle,  sometimes  actually  spurting  out,  and  should  be 


74  METHODS    OF   CULTIVATION   OF    BACTERIA. 

received  in  a  sterile  test-tube.  Several  cubic  centimetres  of 
fluid  can  thus  usually  be  obtained,  no  suction  being  required  ; 
thereafter  it  can  be  examined  bacteriologically  by  the  usual 
methods.  The  depth  of  the  sub-arachnoid  space  from  the  sur- 
face varies  from  a  little  over  an  inch  in  children  to  three  inches, 
or  even  more,  in  adults  —  the  length  of  the  needle  must  be  suited 
accordingly.  In  making  the  puncture  it  is  convenient  to  have 
either  a  sterile  syringe  attached,  or  to  have  the  thick  end  of 
the  needle  covered  with  a  pad  of  sterile  wool,  which  is  of  course 
removed  at  once  when  the  fluid  begins  to  flow. 

The  Bacteriological  Examination  of  Urine.  —  In  such  an 
examination  care  must  be  taken  to  prevent  the  contamination 
of  the  urine  by  extraneous  organisms.  In  the  male  it  is  usually 
sufficient  to  wash  thoroughly  the  glans  penis  and  the  meatus 
with  i-iooo  corrosive  sublimate  —  the  tips  of  the  meatus  being 
everted  for  more  thorough  cleansing.  The  urine  is  then  passed 
into  a  series  of  sterile  flasks,  the  first  of  which  is  rejected  in 
case  contamination  has  occurred.  In  the  female,  after  similar 
precautions  as  regards  external  cleansing,  the  catheter  must  be 
used.  The  latter  must  be  boiled  for  half  an  hour,  and  anointed 
with  olive  oil  sterilised  by  half  an  hour's  exposure  in  a  plugged 
flask  to  a  temperature  of  120°  C.  Here,  again,  it  is  well  to 
reject  the  urine  first  passed.  It  is  often  advisable  to  allow  the 
urine  to  stand  in  a  cool  place  for  some  hours,  to  then  withdraw 
the  lower  portion  with  a  sterile  pipette,  to  centrif  ugalise  this,  and 
to  use  the  urine  in  the  lower  parts  of  the  centrifuge  tubes  for 
microscopic  examination  or  culture. 

Filtration  of  Cultures.  —  For  many  purposes  it  is  necessary 
to  filter  all  the  organisms  from  fluids  in  which  they  may  have 
been  growing.  This  is  especially  done  in  obtaining  the  soluble 
toxic  products  of  bacteria.  The  only  filter  capable  of  keeping 
back  such  minute  bodies  as  bacteria  is  that  formed  from  a  tube 
of  unglazed  porcelain  as  introduced  by  Chamberland.  The 
efficiency  of  such  a  filter  depends  on  the  fineness  of  the  grain 
of  the  clay  from  which  it  is  made  ;  the  finest  is  the  Kitasato  filter 
and  the  Chamberland  "  B "  pattern  ;  the  next  finest  is  the 
Chamberland  "  F "  pattern,  which  is  quite  good  enough  for 
ordinary  work.  There  are  several  filters,  differing  slightly  in 
detail,  all  possessing  the  common  principle.  Sometimes  the 
fluid  is  forced  through  the  porcelain  tube.  In  one  form  the 


FILTRATION    OF   CULTURES. 


75 


filter  consists  practically  of  an  ordinary  tap  screwed  into  the  top 
of  a  porcelain  tube.  Through  the  latter  the  fluid  is  forced  and 
passes  into  a  chamber  formed  by  a  metal  cylinder  which  sur- 
rounds the  porcelain  tube.  The  fluid  escapes  by  an  aperture  at 
the  bottom.  Such  a  filter  is  very  suitable  for  domestic  use,  or 
for  use  in  surgical  operating-theatres.  As  considerable  pressure 
is  necessary,  it  is  evident  it  must  be  put  on  a  pipe  leading 
directly  from  the  main.  Sometimes,  when  fluids  to  be  filtered 
are  very  albuminous,  they  are  forced  through  a  porcelain  cylin- 
der by  compressed  carbonic  acid  gas.  In  ordinary  bacteriologi- 
cal work,  however,  it  is  usually  more  convenient  to  suck  the 
fluid  through  the  porcelain  by  exhausting  the  air  in  the  recepta- 
cle into  which  it  is  to  flow.  This  is  conveniently  done  by  means 
of  a  Geissler's  water-exhaust  pump  (Fig.  38,  g\  which  must  be 
fixed  to  a  tap  leading  directly 
from  the  main.  The  connec- 
tion with  the  tap  must  be 
effected  by  means  of  a  piece 
of  thick-walled  rubber  tubing 
as  short  as  possible,  wired  on 
to  tap  and  pump,  and  firmly 
lashed  externally  with  many 
turns  of  strong  tape.  Before 
lashing  with  the  tape  the 
tube  may  be  strengthened  by 
fixing  round  it  with  rubber 
solution  strips  of  the  rubbered 
canvas  used  for  mending 
punctures  in  the  outer  case 
of  a  bicycle  tire.  A  ma- 
nometer tube  b  and  a  re- 
ceptacle c  (the  latter  to 
catch  any  back  flow  of  water  from  the  pump  if  the  filter  acci- 
dentally breaks)  are  intercepted  between  the  filter  and  the 
pump.  These  are  usually  arranged  on  a  board  a,  as  in  Fig.  38. 
Between  the  tube  /  and  the  pump  g,  and  between  the  tube  d 
and  the  filter,  it  is  convenient  to  insert  lengths  of  flexible  lead 
tubing  connected  up  at  each  end  with  short,  stout-walled  rubber 
tubing. 

Various  modifications  of  the  filter  are  used,  (a)  An  apparatus 


FlG.  38.  —  Geissler's  vacuum-pump  arranged 
with  manometer  for  filtering  cultures.  (The 
tap  and  pump  are  intentionally  drawn  to  a  larger 
scale  than  the  manometer  board  to  show  details.) 


METHODS    OF   CULTIVATION   OF   BACTERIA. 


is  arranged  as  in  Fig.  39.  The  fluid  to  be  filtered  is  placed  in 
the  cylindrical  vessel  a.  Into  this  a  "  candle  "  or  "  bougie  "  of 
porcelain  dips.  From  the  upper  end  of 
the  bougie  a  glass  tube  with  thick  rubber 
connections,  as  in  Fig.  39,  proceeds  to 
flask  b  and  passes  through  one  of  the  two 
perforations  with  which  the  rubber  stop- 
per of  the  flask  is 'furnished.  Through 
the  other  opening  a  similar  tube  proceeds 
to  the  exhaust-pump.  When  the  latter  is 

FIG.   39.  —  Chamberland  s 
candle  and  flask  arranged  for      put  into  action  the  fluid  is  SUCked  through 

the  porcelain  and  passes  over  into  flask  b. 

This  apparatus  is  very  good,  but  not  suitable  for  small  quantities 

of  fluid. 

(b)   A  very  good  apparatus  can  be 

arranged  with  a  lamp  funnel  and  the 

porcelain  bougie.    These  may  be  fitted 

up  in  two  ways,     (i)  An  india-rubber 

washer  is  placed  round  the  bougie  c  at 

its  glazed  end  (vide  Fig.  40).     On  this 

the  narrow  end  of  the  funnel  d,  which 

must,  of  course,  be  of  an  appropriate 

size,  rests.  A  broad  band  of  sheet 
rubber  is  then  wrapped 
round  the  lower  end  of 
the  funnel  and  the  pro- 
jecting part  of  the  bougie.  It  is  firmly  wired  to 
the  funnel  above  and  to  the  bougie  below.  The 
extreme  point  of  the  latter  is  left  exposed,  and 
the  whole  apparatus,  being  supported  on  a  stand, 
is  connected  by  a  glass  tube  with  the  lateral  tube 
of  the  flask  b ;  the  tube  a  is  connected  with  the 
exhaust-pump.  The  fluid  to  be  filtered  is  placed 
between  the  funnel  and  the  bougie  in  the 

S?aCe   '.  and   is   Sufcked   thr0"Sh   int°  the  flask  b- 
rubber  stopper  for  (2)  This  modification  is  shown  in  Fig.  41.     Into 

F^e39.UrP°SeaSin  the    narrow   Part  of   the  funnel  an  india-rubber 
stopper  is  fitted,  which  has  a  perforation  in  it 
sufficiently  large  to  receive  the  candle,  which  it  should  grasp 
tightly. 


FlG.  40.  —  Chamberland's  bou- 
gie arranged  with  lamp  funnel  for 
filtering  a  small  quantity  of  fluid. 


FILTRATION   OF   CULTURES. 


77 


FlG.  42.  —  Muencke's  modification  of 
Chamberland's  filter. 


(c)   Muencke's  modification  of  the  Chamberland  principle  is 

seen  in  Fig.  42.     It  consists  of  a  thick-walled  flask,  a,  the  lower 

part  conical,  the  upper  cylindrical,  with  a  strong  flange  on  the 

lip.    There  are  two  lateral  tubes,  A 

one  horizontal  to   connect  with 

exhaust-pipe,  and    one    sloping, 

by  which  the  contents  may  be 

poured  out.       Passing   into  the 

upper   cylindrical    part    of   the 

flask  is  a  hollow  porcelain  cylin- 
der b,  of 'less  diameter  than  the 

cylindrical  part  of  flask  a.     It  is 

closed  below,  open    above,  and 

rests  by  a  projecting  rim  on  the 

flange  of  the  flask,  an  asbestos 

washer,  c,  being  interposed.    The 

fluid  to  be  filtered  is  placed  in  the  porcelain  cylinder,  and  the 
whole  top  covered,  as  shown  at  /,  with  an  india- 
rubber  cap  with  a  central  perforation  ;  the  tube  d 
is  connected  with  the  exhaust-pump  and  the  tube ' e 
plugged  with  a  rubber  stopper.  When 
a  large  quantity  of  fluid  is  to  be  filtered, 
a  receptacle  such  as  that  shown  in  Fig.  43  may  be 
used.  The  tap  in  its  bottom  enables  the  filtrate 
to  be  removed  without  the  apparatus  being  un- 
shipped, but  it  is  difficult  to  get  the  tap  to  fit  so 
accurately  as  not  to  allow  air  to  pass  into  the 
vacuum  chamber. 

Before  any  one  of  the  above  apparatus  is  used, 
it  ought  to  be  connected  up  as  far  as  possible  and 
sterilised  in  the  Koch's  steriliser.  The  ends  of 
any  important  unconnected  parts  ought  to  have 
pieces  of  cotton  wool  tied  over  them.  After  use 
the  bougie  is  to  be  sterilised  in  the  autoclave,  and 
FiG.43.— Flask  after  being  dried  is  to  be  passed  carefully  through 

fitted  with  porce-  &_ 

lain  bougie  for  fii-  a  Bunsen  flame,  to  burn  off  all  organic  matter.     It 
tering  large  quan-  the  j  tter  is  aiiowec}  to  accumulate,  the  pores  become 

titles  of  fluid. 

filled  up. 

The  success  of  filtration  must  be  tested  by  inoculating  tubes 
of  media  from  the  filtrate,  and  observing  if  growth  takes  place, 


78  METHODS    OF   CULTIVATION    OF    BACTERIA. 

as  there  may  be  minute  perforations  in  the  candles  sufficiently 
large  to  allow  bacteria  to  pass  through.  Filtered  fluids  keep 
for  a  long  time  if  the  openings  of  the  glass  vessels  in  which 
they  are  placed  are  kept  thoroughly  closed,  and  if  these  vessels 
be  kept  in  a  cool  place  in  the  dark.  A  layer  of  sterile  toluol 
about  half  an  inch  thick  ought  to  be  run  on  to  the  top  of 
the  filtered  fluid  to  protect  the  latter  from  the  atmospheric 
oxygen. 

Instead  of  being  filtered  off,  the  bacteria  may  be  killed  by 
various  antiseptics,  chiefly  volatile  oils,  such  as  oil  of  mustard 
(Roux).  These  oils  are  stated  to  have  no  injurious  effect  on  the 
chemical  substances  in  the  fluid,  and  they  may  be  subsequently 
removed  by  evaporation.  It  is  not  practicable  to  kill  the 
bacteria  by  heat  when  their  soluble  products  are  to  be  studied, 
as  many  of  the  latter  are  destroyed  by  a  lower  temperature  than 
is  required  to  kill  the  bacteria  themselves. 

The  Observation  of  Bacterial  Fermentations  in  Sugars. — 
The  capacity  of  certain  species  of  bacteria  to  originate  fermenta- 
tions in  sugars  constitutes  an  important  biological  factor.  The 
end-products  of  such  fermentations  may  be  various.  They 
differ  according  to  the  sugar  employed  and  according  to  the 
species  under  observation,  and  frequently  a  species  which  will 
ferment  one  sugar  has  no  effect  on  another.  The  substances 
finally  produced,  speaking  roughly,  may  be  alcohols,  acids,  or 
gaseous  bodies  (chiefly  carbon  dioxide,  hydrogen,  and  methane). 
For  the  estimation  of  the  two  former  groups  complicated  chem- 
ical procedure  may  be  necessary.  The  formation  of  gases  is, 
however,  usually  taken  as  the  criterion  of  the  possession  of 
fermentative  properties.  Generally  speaking,  it  is  reliable,  and 
the  methods  to  be  pursued  are  simple.  It  must  not  be  forgotten, 
however,  that  some  organisms  give  rise  to  sulphuretted  hydro- 
gen by  breaking  up  the  proteid  present.  The  formation  of  this 
gas  can  be  detected  by  the  blackening  of  lead  acetate  when  it  is 
added  to  the  gas-containing  medium.  The  following  are  the 
chief  methods  for  detecting  the  formation  of  gas :  — 

(i)  Gelatin  Shake  Cultures  (Fig.  44,  a).  — The  gelatin  in  the 
tube  is  melted  as  for  making  plates  ;  while  liquid  it  is  inoculated 
with  the  growth  to  be  observed,  and  shaken  to  distribute  the 
organisms  throughout  the  jelly.  It  is  then  allowed  to  solidify, 
and  is  set  aside  at  a  suitable  temperature.  If  the  bacterium 


BACTERIAL   FERMENTATIONS    IN    SUGARS. 


79 


f- 


^-_ 

1 
'% 

; 

/ 

' 
V 

1 

used  is  a  gas-forming  one,  then,  as  growth  occurs,  little  bubbles 
appear  round  the  colonies.  These  frequently  coalesce  to  form 
bubbles  of  a  larger  size, 
and  those  which  are  super- 
ficial in  process  of  time 
diffuse  out  of  the  medium. 
This  method  is  very  fre- 
quently used  for  studying 
gas  formation  by  B.  coli. 

(2)  Durham's     Tubes 
(Fig.  44,  b).  —  The  plug  of 
a  tube  which  contains  about 
one-third  more  than  usual 
of  a  liquid  medium  is  re- 
moved,  and  a  small  test- 
tube   is    slipped   into   the 
latter   mouth   downwards.         6 
The  plug  is  replaced  and    FIG.  44. 
the  tube    sterilised  thrice 

at  100°  C.  The  air  re- 
maining in  the  smaller  tube 
is  thereby  expelled.  The  tube  is  then  inoculated  with  the  bac- 
terium to  be  tested.  Any  gas  developed  collects  in  the  upper 
part  of  the  inner  tube. 

(3)  The  Fermentation  Tube  (Fig.  44,  c). — This  consists  of  a 
tube  of  the  form  shown,  and  the  figure  also  indicates  the  extent 
to  which  it  ought  to  be  filled.     It  is  inoculated  in  the  bend  with 
the  gas-forming  organism,  and   when   growth  occurs  the  gas 
collects  in  the  upper  part  of  the  closed  limit,  the  medium  being 
displaced  into  the  bulb. 

H.  W.  Hill  has  advantageously  modified  the  ordinary  fer- 
mentation tube  of  Smith  (Fig.  45)  by  having  the  bulb  made  larger 
so  as  to  accommodate  twice  the  quantity  of  fluid  contained  in  the 
branch,  thus  avoiding  wetting  of  the  plugs  during  sterilisation ; 
also,  in  having  replaced  the  sealed  end  of  the  branch  by  a  snugly 
fitting,  hollow,  ground-glass  thimble,  or  stopper,  permitting  one 
to  examine  the  contents  of  the  branch,  either  chemically  or  bac- 
teriologically,  without  contamination  by  fluid  in  the  bulb,  such  as 
happens  when  using  the  ordinary  form  of  the  tube.  In  carrying 
out  this  examination  it  is  obviously  necessary  to  first  replace 


Tubes  for  demonstrating  gas-formation 
by  bacteria. 

a,  tube  with  "  shake  "  culture. 

b,  Durham's  fermentation  tube. 

c,  ordinary  form  of  fermentation  tube  (Smith's). 


8o 


METHODS    OF   CULTIVATION    OF   BACTERIA. 


the  cotton  plug  by  a  sterile  rubber  stopper  before  opening  up 
the  closed  arm. 

The  composition  of  the  medium  is,  of  course,  of  great  impor- 
tance, and  in  testing  the  effect  of  a  bacterium  on  a  given  sugar 
it  is  essential  that  this  sugar  alone  be  present. 
The  first  method  is  usually  used  with  ordinary 
gelatin,  and  the  gas-formation  in  most  cases 
results  from  fermentation  of  the  glucose 
naturally  present  in  the  medium  from  trans- 
formation of  the  glycogen  of  muscle.  (It  is 
only  a  more  delicate  method  of  demonstrating 
what  sometimes  occurs  along  the  line  of 
growth  in  an  ordinary  gelatin  stab-culture.) 
The  amount  of  glucose  naturally  present, 
however,  varies  much,  and  therefore  glucose 
should  be  added  to  the  medium  if  the  effects 
on  this  sugar  are  to  be  observed.  When 
other  sugars  —  lactose,  mannite,  etc.  —  are  to 
FIG.  45.— mil's  modi-  be  tested,  these  should  be  added  either  to  a 
fication  of  Smith's  fer-  simple  peptone  solution  as  Durham  recom- 

mentation  tube.  ,  .         ... 

mends,  or  to  bouillon  previously  treed  from 
dextrose  as  described  below  —  fermentation  being  observed  by 
either  of  the  methods  (2)  or  (3). 

To  obtain  a  "  dextrose-free  "  bouillon,  Smith  advises  that  the  beef  infusion, 
prepared  by  extracting  in  the  cold  or  at  60°  C.  for  twelve  hours,  be  inoculated 
in  the  evening  with  a  rich  fluid  culture  of  B.  coli  and  placed  in  the  thermostat 
over  night.  Early  the  following  morning  the  infusion,  covered  with  a  layer  of 
froth,  is  boiled,  filtered,  peptone  and  salt  added,  and  neutralisation  and  sterili- 
sation carried  out  as  usual.  As  a  test  for  the  complete  removal  of  the  sugar, 
a  fermentation  tube  of  the  broth  when  inoculated  with  B.  coli  will  no  longer 
give  a  growth  in  the  closed  arm  of  the  tube,  the  fluid  there  remaining  perfectly 
clear.  When  the  various  sugars  are  added  to  such  a  broth,  it  is  strongly 
advised  to  sterilise  by  the  intermittent  method,  for  the  heat  of  the  autoclave 
is  almost  sure  to  produce  chemical  changes  in  the  sugars  through  the  presence 
of  alkali  and  other  constituents  in  the  medium. 

The  Observation  of  Indol  formation  by  Bacteria.  —  The  for- 
mation of  indol  from  albumin  by  a  bacterium  sometimes  con- 
stitutes an  important  specific  characteristic.  To  observe  indol 
production  the  bacterium  is  grown  preferably  at  incubation  tem- 
perature on  a  fluid  medium  containing  peptone.  The  latter  may 
either  be  ordinary  dextrose-free  bouillon  or  peptone  solution 


OBSERVATION   OF   INDOL   FORMATION.  8l 

(see  p.  43).  Indol  production  is  recognised  by  the  fact  that 
when  acted  on  by  nitric  acid  in  the  presence  of  nitrites,  a  nitroso- 
indol  compound  is  produced,  which  has  a  rosy  red  colour.  Some 
bacteria  (e.g.  the  cholera  vibrio)  produce  nitrites  as  well  as  indol, 
but  often,  in  making  the  test  (e.g.  in  the  case  of  B.  coli),  the 
nitrites  must  be  added.  This  may  be  effected  by  using  yellow 
nitric  acid  (which  of  course  contains  nitrous  acid)  for  the  test, 
or  by  adding  to  an  ordinary  tube  of  medium  i  c.c.  of  a  .02  per 
cent  solution  of  potassium  nitrite,  and  testing  with  pure  nitric 
or  sulphuric  acid.  In  any  case  only  a  drop  of  the  acid  need  be 
added  to  say  10  c.c.  of  medium.  If  no  result  be  obtained  at 
once,  it  is  well  to  allow  the  tube  to  stand  for  an  hour,  as  some- 
times the  reaction  is  very  slowly  produced.  The  amount  of  indol 
produced  by  a  bacterium  seems  to  vary  very  much  with  certain 
unknown  qualities  of  the  peptone,  and  differences  in  ability  to 
form  indol  from  a  given  sample  of  peptone  are  noticeable,  too, 
in  races  of  bacteria  of  the  same  species.  It  is  well  therefore 
to  test  a  series  of  peptones  with  an  organism  (such  as  the  B. 
coli)  known  to  produce  indol,  and  noting  the  sample  with  which 
the  best  reaction  is  obtained,  to  reserve  it  for  making  media  to 
be  used  for  the  detection  of  this  product. 

The  Drying  of  Substances  in  vacuo.  —  As  many  substances, 
for  example  toxins  and 
antitoxins,  with  which 
bacteriology  is  con- 
cerned would  be  de- 
stroyed by  drying  with 
heat  as  is  done  in  or- 
dinary chemical  work, 
it  is  necessary  to  re- 
move the  water  at  the 
ordinary  room  tem- 
perature. This  is  most 
quickly  effected  by 
drying  in  vacuo  in  the 
presence  of  some  sub- 
stance such  as  strong 

.     ,  .  ,  FIG.  46.  —  Gervk's  air-pump  for  drying  m  vacuo. 

sulphuric    acid  which 

readily  takes  up  water  vapour.     The  vacuum    produced   by  a 

water-pump  is  here  not  available,  as  in  such  a  vacuum  there 


82  METHODS   OF   CULTIVATION   OF   BACTERIA. 

must  always  be  water  vapour  present.  An  air-pump  is  there- 
fore to  be  employed.  Here  we  have  found  the  Geryk  pump 
most  efficient,  and  it  has  this  further  advantage,  that  its 
internal  parts  are  lubricated  with  an  oil  of  very  low  vapour 
density  so  that  almost  a  perfect  vacuum  is  obtainable.  The 
apparatus  is  shown  in  Fig.  46.  The  vacuum  chamber  consists 
of  a  bell-jar  set  on  a  brass  plate.  A  perforation  in  the  centre 
of  the  latter  leads  into  the  pipe  a,  which  can  be  connected  by 
strong  walled  rubber  tubing  with  the  air-pump,  and  which 
can  be  cut  off  from  the  latter  by  a  stopcock  b.  In  using 
the  apparatus  the  substance  to  be  dried  is  poured  out  in  flat 
dishes  (one-half  of  a  Petri  dish  does  very  well),  and  these  are 
stacked  alternately  with  similar  dishes  of  strong  sulphuric  acid  on 
a  stand  which  rests  on  the  brass  plate.  The  edge  of  the  bell-jar 
is  well  luted  with  unguentum  resinae  and  placed  in  position  and 
the  chamber  exhausted.  In  a  few  hours,  if,  as  is  always  advis- 
able, each  dish  have  contained  only  a  thin  layer  of  fluid,  the 
drying  will  be  complete.  The  vacuum  is  then  broken  by  ad- 
mitting air  very  slowly  through  a  bye-pass  c,  and  the  bell-jar  is 
removed.  In  such  an  apparatus  it  is  always  advisable,  as  is 
shown  in  the  figure,  to  have  interposed  between  the  pump  and 
the  vacuum  chamber  a  Wolff's  bottle  containing  sulphuric  acid. 
This  protects  the  oil  of  the  pump  from  contamination  with  water 
vapour.  Whenever  the  vacuum  is  produced  the  rubber  tube 
should  be  at  once  disconnected  from  a,  the  cock  b  being  shut. 
It  is  advisable  when  the  apparatus  is  exhausted  to  cover  the 
vacuum  chamber  and  the  Wolff's  bottle  with  wire  guards  covered 
with  strong  cloth  in  case,  under  the  external  pressure,  the  glass 
vessels  give  way. 

The  Storing  and  Incubation  of  Cultures.  —  Gelatin  cultures 
must  be  grown  at  a  temperature  below  their  melting-point,  i.e. 
for  10  per  cent  gelatin,  below  22°  C.  They  are  usually  kept  in 
ordinary  rooms,  which  vary,  of  course,  in  temperature  at  different 
times,  but  which  have  usually  a  range  of  from  about  12°  C.  to 
1 8°  C.  Agar  and  serum  media  are  usually  employed  to  grow 
bacteria  at  a  higher  temperature,  corresponding  to  that  at  which 
the  organisms  grow  best,  usually  37°  C.  in  the  case  of  pathogenic 
organisms.  For  the  purpose  of  maintaining  a  uniform  tem- 
perature incubators  are  used.  These  vary  much  in  the  details 
of  their  structure,  but  all  consist  of  a  chamber  with  double  walls 


STORING   AND    INCUBATION    OF   CULTURES. 


between  which  some  fluid  (water  or  glycerin  and  water)  is  placed, 
which,  when  raised  to  a  certain  temperature,  ensures  a  fairly 
constant  distribution  of  the  heat  round  the  y 

chamber.  The  latter  is  also  furnished  with  ...--••""••••-... 
double  doors,  the  inner  being  usually  of  glass. 
Heat  is  supplied  from  a  burner  fixed  below. 
These  burners  vary  much  in  design.  Some- 
times a  mechanism  devised  in  Koch's  laboratory 
is  affixed,  which  automatically  turns  off  the  gas 
if  the  light  be  accidentally  extinguished.  Be- 
tween the  tap  supplying  the  gas,  and  the  burner, 
is  interposed  a  gas  regulator.  Such  regulators 
vary  in  design,  but  for  ordinary  chambers  which 
require  to  be  kept  at  a  constant  temperature, 
Reichert's  is  as  good  and  simple  as  any  and 
is  not  expensive.  It  is  shown  in  Fig.  47. 

It  consists  of  a  long  tube  /  closed  at  the  lower  end, 
open  at  the  upper,  and  furnished  with  two  lateral  tubes. 
The  lower  part  is  filled  with  mercury  up  to  a  point  above  FIG.  47.  —  Reichert's 
the  level  of  the  lower  lateral  tube.  The  end  of  the  latter  is  «as  resulator- 
closed  by  a  brass  cap  through  which  a  screw  d  passes,  the  inner  end  of  which 
lies  free  in  the  mercury.  The  height  of  the  latter  in  the  perpendicular  tube 
can  thus  be  varied  by  increasing  or  decreasing  the  capacity  of  the  lateral  tube 
by  turning  the  screw  a  few  turns  out  of  or  into  it.  Into  the  upper  open  end 
of  the  perpendicular  tube  fits  accurately  a  bent  tube,  g,  drawn  out  below  to  a 
comparatively  small  open  point,  <:,  and  having  in  its  side  a  little  above  the  point 
a  minute  needle-hole  called  the  peephole  or  bye-pass  e.  To  fix  the  apparatus 
the  long  mercury  bulb  is  placed  in  the  jacket  of  the  chamber  to  be  controlled, 
tube  a  is  connected  to  gas  supply,  tube  b  with  the  burner.  The  upper  level 
of  the  mercury  should  be  some  distance  below  the  lower  open  end  of  tube  c. 
The  burner  is  now  lit.  The  gas  passes  in  at  a  through  c  and  e  and  out  at  b  to 
the  burner.  When  the  thermometer  in  the  interior  of  the  chamber  indicates 
that  the  desired  temperature  has  been  reached,  the  screw  d  is  turned  till  the 
mercury  reaches  the  end  of  the  tube  c.  Gas  can  only  now  pass  through  the 
peephole  ^,  and  the  flame  goes  down.  The  contents  of  the  jacket  cool,  the  mer- 
cury contracts  off  the  end  of  tube  c,  and  the  flame  rises.  This  alternation 
going  on,  the  temperature  of  the  chamber  is  kept  very  nearly  constant.  If  the 
mercury  cuts  off  the  gas  supply  before  the  desired  temperature  is  reached,  and 
the  screw  d  is  as  far  out  as  it  will  go,  then  some  of  the  mercury  must  be 
removed.  Similarly,  if  when  the  desired  temperature  is  reached  and  the  screw 
d  is  as  far  in  as  it  can  go,  the  mercury  does  not  reach  c,  some  more  must  be 
introduced.  If  the  amount  of  gas  which  passes  through  the  peephole  is  suf- 
ficient still  to  raise  the  temperature  of  the  chamber  when  c  is  closed  by  the 
rise  of  the  mercury,  then  the  peephole  is  too  large.  Tube  c  must  be  unshipped 


84 


METHODS    OF   CULTIVATION   OF   BACTERIA. 


and  e  plastered  over  with  sealing-wax,  which  is  pricked,  while  still  soft,  with  a 
very  fine  needle.  The  gas  flame,  when  only  the  peephole  is  supplying  gas, 
ought  to  be  sufficiently  large  not  to  be  blown  out  by  small  currents  of  air.  If 
the  pressure  of  gas  supplied  to  a  regulator  varies  much  in  the  24  hours  a  press- 
ure regulator  ought  to  be  interposed  between  the  gas-tap  and  the  instrument. 
Several  varieties  of  these  can  be  obtained.  In  all  cases  g  ought  to  be  fixed  to 
b  with  a  turn  of  wire.  Greater  accuracy  in  regulation  can  be  obtained  if  some 
liquid  paraffin  ("  albolene  ")  is  deposited  upon  the  surface  of  the  mercury  to  the 
depth  of  3  or  4  mm. 

The  varieties  of  incubators  are,  as  we  have  said,  numerous. 
The  most  complicated  and  expensive    are   made   by  German 

manufacturers.  Many  of 
these  are  unsatisfactory. 
They  easily  get  out  of  order 
and  are  difficult  to  repair. 
We  have  found  those  of 
Hearson,  of  London,  ex- 
tremely good,  and  in  pro- 
portion to  their  size  much 
cheaper  than  the  German 
articles, 
with  an 

lator.  In  America,  manu- 
facturers will  be  found  who 
can  supply  incubators  the 
equal  of  any  of  the  foreign 

FIG.  48.  —  Hearson  s  incubator  lor  use  at  37°  C.  & 

makes  on  the  market.    It  is 

preferable  in  using  an  incubator  to  connect  the  regulator  with  the 
gas  supply  and  with  the  Bunsen  by  flexible  metal  tubing.  It  is 
necessary  to  see  that  there  is  not  too  much  evaporation  from  the 
surface  of  cultures  placed  within  incubators,  otherwise  they  may 
quickly  dry  up.  It  is  thus  advisable  to  raise  the  amount  of  water 
vapour  in  the  interior  by  having  in  the  bottom  of  the  incubator  a  flat 
dish  full  of  water,  from  which  evaporation  may  take  place.  Tubes 
which  will  require  to  be  long  in  the  incubator  should  have  their 
plugs  covered  either  by  india-rubber  caps  or  by  pieces  of  sheet  rub- 
ber tied  over  them.  These  caps  should  be  previously  sterilised  in 
i-iooo  corrosive  sublimate,  and  then  dried.  Before  they  are 
placed  on  the  tubes  the  cotton-wool  plug  ought  to  be  well  singed 
in  a  flame.  Or  plugs  may  be  impregnated  with  paraffin  applied 
at  boiling  heat,  which  thoroughly  prevents  the  growth  of  moulds 


They    are    fitted 
admirable    regu- 


GENERAL   LABORATORY   RULES.  85 

or  bacteria  through  the  cotton  and  the  escape  of  water  by  evap- 
oration. "  Cool  "  incubators  are  often  used  for  incubating  gelatin 
at  21°  to  22°  C.  An  incubator  of  this  kind,  fitted  with  a  low- 
temperature  Hearson's  regulator,  is  very  satisfactory. 

Permanent  Preservation  of  Cultures.  —  This  may  be  conven- 
iently effected  by  means  of  formalin  vapour.  The  cotton  wool 
of  the  tube  containing  the  culture  to  be  preserved  is  removed 
and  soaked  in  formalin  (40  per  cent  formic  aldehyde).  It  is 
then  replaced  and  covered  with  an  india-rubber  cap.  After 
exposure  to  the  vapour  in  this  way  for  two  or  three  days,  the 
culture  will  be  found  to  be  dead.  The  final  stage  in  the  process 
is  to  close  the  open  end  of  the  tube  so  as  to  prevent  evaporation. 
Melted  sealing-wax  or  other  substance  may  be  poured  over  the 
cotton  wool,  which  is  first  burned  off  down  to  the  tube,  the  whole 
being  then  covered  by  an  india-rubber  cap,  or  the  upper  end  of 
the  tube  may  be  melted  in  a  Bunsen  flame,  and  thus  sealed.  In 
the  latter  case,  tubes  longer  than  those  generally  employed 
should  be  used,  so  as  to  leave  a  longer  portion  at  the  top  beyond 
the  medium,  otherwise  in  sloped  tubes  part  of  the  medium  is  apt 
to  be  melted.  Cultures  preserved  in  this  way  maintain  their 
form  practically  unchanged  for  several  years,  though  many  col- 
oured growths  are  apt  to  lose  the  colour.  Liquefied  gelatin 
usually  becomes  solidified  by  the  action  of  formalin  vapour,  so 
that  the  tubes  can  be  freely  handled.  In  the  case  of  agar  tubes, 
any  water  of  condensation  should  first  of  all  be  carefully  poured 
off. 

General  Laboratory  Rules.  —  On  the  working  bench  of  every 
bacteriologist  there  should  be  a  large  dish  of  i-iooo  solution  of 
mercuric  chloride  in  water.  Into  this,  all  tubes,  vessels,  plates, 
hanging-drop  cultures,  etc.,  which  have  contained  bacteria,  and 
with  which  he  has  finished,  ought  to  be  at  once  plunged  (in  the 
case  of  tubes,  the  tube  and  plug  should  be  put  in  separately). 

On  no  account  whatever  are  such  infected  articles  to  be  left 
lying  about  the  laboratory.  The  basin  is  to  be  repeatedly 
cleaned  out.  All  the  glass  is  carefully  washed  in  repeated 
changes  of  tap  water  to  remove  the  last  trace  of  perchloride  of 
mercury,  a  very  minute  quantity  of  which  is  sufficient  to  inhibit 
growth.  Old  cultures  which  have  been  stored  for  a  time  and 
from  which  fresh  sub-cultures  have  been  made  ought  to  be 
steamed  in  the  Koch  steriliser  for  two  or  three  hours,  or  in  the 


86  METHODS   OF   CULTIVATION    OF   BACTERIA. 

autoclave  for  a  shorter  period,  and  the  tubes  thoroughly  washed 
out.  Besides  a  basin  of  mercuric  chloride  solution  for  infected 
apparatus,  etc.,  there  ought  to  be  a  second  reserved  for  the 
worker's  hands  in  case  of  any  accidental  contamination.  When, 
as  in  public  health  work,  a  large  number  of  tubes  are  being  daily 
put  out  of  use,  they  may  be  put  into  an  enamelled  slop-pail,  and 
this  when  full  is  placed  in  the  steam  steriliser. 

A  white  glazed  tile  on  which  a  bell-jar  can  be  set  is  very  con- 
venient to  have  on  a  bench.  Infective  material  in  watch-glasses 
can  be  placed  thus  under  cover  while  investigation  is  going  on, 
and  if  anything  is  spilled  the  whole  can  be  easily  disinfected. 
In  making  examinations  of  organs  containing  virulent  bacteria, 
the  hands  should  be  previously  dipped  in  i-iooo  mercuric 
chloride  and  allowed  to  remain  wet  with  this  lotion.  No  food 
ought  to  be  partaken  of  in  the  laboratory,  and  pipes,  etc.,  are 
not  to  be  laid  with  their  mouthpieces  on  the  bench.  No  label 
is  to  be  licked  with  the  tongue.  Before  leaving  the  laboratory 
the  bacteriologist  ought  to  wash  the  hands  and  forearms  with 
i-iooo  mercuric  chloride  and  then  with  yellow  soap.  In  the 
case  of  ary  fluid  containing  bacteria  being  accidentally  spilt  on 
the  bench  or  floor,  i-iooo  mercuric  chloride  is  to  be  at  once 
poured  on  the  spot.  The  air  of  the  laboratory  ought  to  be  kept 
as  quiet  as  possible. 


CHAPTER   III. 

MICROSCOPIC    METHODS  — GENERAL   BACTERIOLOGICAL 
DIAGNOSIS  — INOCULATION   OF   ANIMALS. 

The  Microscope.  —  For  ordinary  bacteriological  work  a  good 
microscope  is  essential.  It  ought  to  have  a  heavy  stand,  with 
rack  and  pinion  and  fine  adjustment,  a  double  mirror  (flat  on 
one  side,  concave  on  the  other),  a  good  condenser,  with  an  iris 
diaphragm,  and  a  triple  nose-piece.  It  is  best  to  have  three 
objectives,  either  Zeiss  A,  D,  and  ^  inch  oil  immersion,  or  the 
lenses  of  other  makers  corresponding  to  these.  The  oil  immer- 
sion lens  is  essential.  It  is  well  to  have  two  eyepieces,  say 
Nos.  2  and  4  of  Zeiss  or  lenses  of  corresponding  strengths. 
The  student  must  be  thoroughly  familiar  with  the  focussing 
of  the  light  on  the  lens  by  means  of  the  condenser,  and  also 
with  the  use  of  the  immersion  lens.  It  may  here  be  remarked 
that  when  it  is  desired  to  bring  out  in  sharp  relief  the  mar- 
gins of  unstained  objects,  e.g.  living  bacteria  in  a  fluid,  a 
narrow  aperture  of  the  diaphragm  should  be  used,  whereas, 
in  the  case  of  stained  bacteria,  when  a  pure  colour  picture  is 
desired,  the  diaphragm  ought  to  be  widely  opened.  The  flat 
side  of  the  mirror  ought  to  be  used  along  with  the  condenser. 
When  the  observer  has  finished  for  the  time  being  with  the 
immersion  lens  he  ought  to  wipe  off  the  oil  with  a  piece  of 
silk  or  very  fine  washed  linen.  If  the  oil  has  dried  on  the 
lens  it  may  be  moistened  with  xylol  —  never  with  alcohol, 
which  will  dissolve  the  material  by  which  the  lens  is  fixed  in 
its  metal  carrier. 

Microscopic  Examination  of  Bacteria,  i.  Hanging-drop  Pre- 
parations.—  Micro-organisms  may  be  examined:  (i)  alive  or 
dead  in  fluids;  (2)  in  film  preparations;  (3)  in  sections  of 
tissues.  In  the  two  last  cases  advantage  is  always  taken  of  the 
affinity  of  bacteria  for  certain  stains.  When  they  are  to  be 
examined  in  fluids  a  drop  of  the  liquid  may  be  placed  on  a  slide 

87 


88  MICROSCOPIC   METHODS. 

and  covered  with  a  cover-glass.1  It  is  more  usual,  however,  to 
employ  hanging-drop  preparations.  The  technique  of  making 
these  has  already  been  described  (p.  68).  In  examining  them 
microscopically,  it  is  necessary  to  use  a  very  small  diaphragm- 
It  is  best  to  focus  the  edge  of  the  drop  with  a  low-power  objec- 
tive, and,  arranging  the  slide  so  that  part  of  the  edge  crosses 
the  centre  of  the  field,  to  clamp  the  preparation  in  this  position. 
A  high-power  lens  is  then  turned  into  position  and  lowered  by 
the  coarse  adjustment  to  a  short  distance  above  its  focal  distance  ; 
it  is  now  carefully  screwed  down  by  the  fine  adjustment,  the  eye 
being  kept  at  the  tube  meanwhile.  The  shadow  of  the  edge  will 
be  first  recognised,  and  then  the  bacteria  must  be  carefully 
looked  for.  Often  a  dry  lens  is  sufficient,  but  for  some  purposes 
the  oil  immersion  is  required.  If  the  bacteria  are  small  and 
motile  a  beginner  may  have  great  difficulty  in  seeing  them,  and 
it  is  well  to  practise  at  first  on  some  large  non-motile  form  such 
as  anthrax.  In  fluid  preparations  the  natural  appearance  of 
bacteria  may  be  studied,  and  their  rate  of  growth  determined. 
The  great  use  of  such  preparations,  however,  is  to  find  whether 
or  not  the  bacteria  are  motile,  and  for  determining  this  point 
it  is  advisable  to  use  either  broth  or  agar  cultures  not  more  than 
twenty-four  hours  old.  In  the  latter  case  a  small  fragment  of 
growth  is  broken  down  in  broth  or  in  sterile  water.  Sometimes 
it  is  an  advantage  to  colour  the  solution  in  which  the  hanging- 
drop  is  made  up  with  a  minute  quantity  of  an  aniline  dye,  say 
a  small  crystal  of  gentian-violet  to  100  c.c.  of  bouillon.  Such 
a  degree  of  dilution  will  not  have  any  effect  on  the  vitality  of 
the  bacteria.  Ordinarily,  living  bacteria  will  not  take  up  a  stain, 
but  even  though  they  do  not,  the  contrast  between  the  unstained 
bacteria  and  the  tinted  fluid  will  enable  the  observer  more  easily 
to  recognise  them. 

2.  Film  Preparations,  (a)  Dry  Method.  —  This  is  the  most 
extensively  applicable  method  of  microscopically  examining  bac- 
teria. Fluids  containing  bacteria,  such  as  blood,  pus,  scrapings 
of  organs,  can  be  thus  investigated,  as  also  cultures  in  fluid  and 
solid  media.  The  first  requisite  is  a  perfectly  clean  cover-glass. 
Many  methods  are  recommended  for  obtaining  such.  The  test 

1  In  bacteriological  work  it  is  essential  that  cover-glasses  of  No.  I  thickness 
(i.e.  .14  mm.  thick)  should  be  used,  as  those  of  greater  thickness  are  not  suitable  for 
a  j%  in.  lens. 


FILM   PREPARATIONS.  89 

of  this  being  accomplished  is  that,  when  the  drop  of  fluid  con- 
taining the  bacteria  is  placed  upon  the  glass,  it  can  be  uniformly 
spread  with  the  platinum  needle  all  over  the  surface  without 
showing  any  tendency  to  retract  into  droplets. 

The  best  method  to  pursue  is  to  keep  the  cover-slips  in  a 
vessel  containing  95  per  cent  alcohol.  When  required  for  use  a 
slip  is  taken  and  polished  with  a  piece  of  old  linen  or  cotton  cloth, 
which  must  be  perfectly  clean ;  then  placing  the  cover-slip  in 
the  forceps,  pass  it  through  the  flame 
six  or  eight  times,  not  too  slowly  so 
as  to  risk  destroying  it,  and  it  will  be 
found  that  the  lowermost  surface  will  FlG-  49-  —  Comet's  forceps  for  hoid- 

.  .        .  ing  cover-glasses. 

be  perfectly  free   from   any  greasy 

substance.  Another  method  is  that  recommended  by  Van 
Ermengem.  The  cover-glasses  are  placed  for  some  time  in  a 
mixture  of  concentrated  sulphuric  acid  6  parts,  potassium  bichro- 
mate 6  parts,  water  100  parts,  then  washed  thoroughly  in  water 
and  stored  in  absolute  alcohol.  For  use,  a  cover-glass  is  either 
dried  by  wiping  with  a  clean  duster  or  is  simply  allowed  to  dry. 
This  method  will  amply  repay  the  trouble,  and  really  saves  time 
in  the  end.  A  clean  cover  having  been  obtained,  the  film  pre- 
paration can  now  be  made.  If  a  fluid  is  to  be  examined,  a 
loopful  may  be  placed  on  the  cover-glass,  and  either  spread  out 


FlG.  50.  —  Stewart's  cover-glass  forceps. 

over  the  surface  with  the  needle,  or  another  clean  cover  may  be 
placed  on  the  top  of  the  first,  the  drop  thus  spread  out  between 
them  and  the  two  then  drawn  apart.  When  a  culture  on  a  solid 
medium  is  to  be  examined  a  loopful  of  distilled  water  is  placed 
on  the  cover-glass  and  a  minute  particle  of  growth  rubbed  up  in 
it  and  spread  over  the  glass.  The  great  mistake  made  by  be- 
ginners is  to  take  too  much  of  the  growth.  The  point  of  the 
straight  needle  should  just  touch  the  surface  of  the  culture,  and 
when  this  is  rubbed  up  in  the  droplet  of  water  and  the  film 
dried,  there  should  be  an  opaque  cloud  just  visible  on  the  cover- 
glass.  When  the  film  has  been  spread  it  must  next  be  dried  by 


90  MICROSCOPIC   METHODS. 

being  waved  backwards  and  forwards  at  arm's  length  above  a 
Bunsen  flame.  The  film  must  then  be  fixed  on  the  glass  by 
being  passed  four  or  five  times  quickly  through  the  flame.  In 
doing  this  a  good  plan  is  to  hold  the  cover-glass  between  the 
right  forefinger  and  thumb  ;  if  the  fingers  just  escape  being 
burned  no  harm  will  accrue  to  the  bacteria  in  the  film. 

In  making  films  of  a  thick  fluid  such  as  pus  it  is  best  to 
deposit  a  small  quantity  centrally  on  the  cover-slip,  then  to  place 
another  cover  on  top  and  draw  the  two  apart.  The  result  will 
be  a  film  of  uniform  depth  throughout,  available  at  almost 
all  parts  for  examination.  Scrapings  of  organs  are  very  con- 
venient if  only  the  presence  or  absence  of  organisms  is  inquired 
after.  Such  scrapings  may  be  smeared  directly  on  the  cover- 
glasses. 

In  the  case  of  blood,  a  fairly  large  drop  should  be  allowed 
to  spread  itself  between  two  cover-glasses,  which  are  then  to 
be  slipped  apart,  and  being  held  between  the  forefinger  and 
thumb  are  to  be  dried  by  a  rapid  to-and-fro  movement  in  the 
air.  A  film  prepared  in  this  way  may  be  too  thick  at  one 
edge,  but  at  the  other  is  beautifully  thin.  If  it  is  desired 
to  preserve  the  red  blood  corpuscles .  in  such  a  film  it  may 
be  fixed  by  one  of  the  following  methods :  by  being  placed 
(a)  in  a  hot-air  chamber  at  120°  C.  for  half  an  hour;  (£)  in 
a  mixture  of  equal  parts  of  alcohol  and  ether  for  half  an  hour, 
then  washed  and  dried  ;  (c)  in  formol-alcohol  (Gulland)  (formalin 
i  part,  absolute  alcohol  9  parts)  for  five  minutes,  then  washed 
and  dried ;  or  (d)  in  a  saturated  solution  of  corrosive  sublimate 
for  two  or  three  minutes,  then  washed  well  in  running  water 
and  dried.  (Fig.  78  shows  a  film  prepared  by  the  last  method.) 
In  the  case  of  urine,  the  specimen  must  be  allowed  to  stand, 
and  films  made  from  any  deposit  which  occurs ;  or,  what  is 
still  better,  the  urine  is  centrifugalised,  and  films  made  from 
the  deposit  which  forms.  After  dried  films  are  thus  made 
from  urine  it  is  an  advantage  to  place  a  drop  of  distilled  water 
on  the  film  and  heat  gently  to  dissolve  the  deposit  of  salts ;  then 
wash  in  water  and  dry.  In  this  way  a  much  clearer  picture  is 
obtained  when  the  preparation  is  stained.  Preparations  of  broth 
or  milk-cultures  can  be  rendered  free  of  stain-retaining  material 
by  allowing  glacial  acetic  acid  to  act  upon  the  film,  after  fixation, 
for  five  seconds,  and  then  washing  thoroughly  in  water. 


EXAMINATION   OF   BACTERIA   IN   TISSUES.  91 

Films  dried  and  fixed  by  the  above  methods  are  now  ready 
to  be  stained  by  the  methods  to  be  described  below. 

(b]  Wet   Method.  —  If   it   is    desired    to    examine    the   fine 
histological  structure  of  the  cells  of   a  discharge   as  well  as 
to  investigate  the  bacteria  present,  it  is  advisable  to  substitute 
"wet"  films  for  the  "dried"  films,  the  preparation  of  which 
has  been  described.      The   nuclear    structure,   mitotic   figures, 
etc.,   are   by  this    method  well   preserved,   whereas    these  are 
considerably   distorted   in   dried   films.      The    initial    stages    in 
the  preparation  of  wet  films  are  the  same  as  above,  but  instead 
of  being  dried  in  air  they  are  placed,  while  still  wet,  film  down- 
wards in  the  fixative.     The  following  are  some  of  the  best  fixing 
methods  :  — 

(a}  A  saturated  solution  of  perchloride  of  mercury  in  .75  per  cent  sodium 
chloride  ;  fix  for  five  minutes.  Then  place  the  films  for  half  an  hour,  with 
occasional  gentle  shaking,  in  .75  per  cent  sodium  chloride  solution  to  wash 
out  the  corrosive  sublimate  ;  they  are  thereafter  washed  in  successive  strengths 
of  methylated  spirit.  After  this  treatment  the  films  are  stained  and  treated  as 
if  they  were  sections. 

(£)  Formol-alcohol  —  formalin  i  part,  absolute  alcohol  9.  Fix  films  for 
three  minutes ;  then  wash  well  in  methylated  spirit.  This  is  an  excellent 
and  very  rapid  method. 

(c )  Another  excellent  method  of  fixing  has  been  devised  by  Gulland.    The 
fixing  solution  has  the  composition  —  absolute  alcohol,  25  c.c.,  pure  ether, 
25  c.c.,  alcoholic  solution  of  corrosive  sublimate  (2  grm.  in  10  c.c.  of  alcohol), 
about  5   drops.     The  films  are  placed  in  this  solution  for  five  minutes  or 
longer.     They  are  then  .washed  well  in  water,  and  are  ready  for  staining.     A 
contrast  stain  can  be  applied  at  the  same  time  as  the  fixing  solution,  by  satu- 
rating the  25  c.c.  of  alcohol  with  eosin  before  mixing.    Thereafter  the  bacteria, 
etc.,  may  be  stained  with  methylene-blue  or  other  stain,  as  described  below. 
This  method  has  the  advantage  over  (<z)  that,  as  a  small  amount  of  corrosive 
sublimate  is  used,  less  washing  is  necessary  to  remove  it  from  the  preparation, 
and  deposits  are  less  liable  to  occur. 

3.  Examination  of  Bacteria  in  Tissues.  —  For  the  examina- 
tion of  bacteria  in  the  tissues,  the  latter  must  be  fixed  and 
hardened,  in  preparation  for  being  cut  with  a  microtome. 
Fixation  consists  in  so  treating  a  tissue  that  it  shall  permanently 
maintain,  as  far  as  possible,  the  condition  it  was  in  when  re- 
moved from  the  body.  Hardening  consists  in  giving  such  a 
fixed  tissue  sufficient  consistence  to  enable  a  thin  section  of  it 
to  be  cut.  A  tissue,  after  being  hardened,  may  be  cut  in  a 
freezing  microtome,  but  far  finer  results  can  be  obtained  by 


92  MICROSCOPIC   METHODS. 

embedding  the  tissue  in  solid  paraffin  and  cutting  with  some 
of  the  more  delicate  microtomes  of  which,  for  pathological 
purposes,  the  small  Cambridge  rocker  is  by  far  the  best.  For 
bacteriological  purposes  embedding  in  celloidin  is  not  advisable, 
as  the  celloidin  takes  on  the  aniline  dyes  which  are  used  for 
staining  bacteria,  and  is  apt  thus  to  spoil  the  preparation,  and 
besides  thinner  sections  can  be  obtained  by  the  paraffin  method. 
The  Fixation  and  Hardening  of  Tissues.  —  The  following 
are  amongst  the  best  methods  for  bacteriological  purposes :  — 

(a)  Absolute  alcohol  may  be  used  for  the  double  purpose  of  fixing  and 
hardening.     If  the  piece  of  tissue  is  not  more  than  \  inch  in  thickness  it  is 
sufficient  to  keep  it  in  this  reagent  for  one  or  two  days.     If  the  pieces  are 
thicker  a  longer  exposure  is  necessary,  and  in  such  cases  it  is  better  to  change 
the  alcohol  at  the  end  of  the  first  twenty-four  hours.     The  tissue  must  be 
tough  without  being  hard,  and  the  necessary  consistence,  as  estimated  by 
feeling  with  the  fingers,  can  only  be  judged  of  after  some  experience.     If 
the   tissues  are  not  to  be  cut  at  once,  they  may  be  preserved  in  50  per 
cent  spirit. 

(b)  Formol-alcohol — formalin  I,  absolute  alcohol  9.     Fix  for  not  more 
than  twenty-four  hours ;  then  place  in  absolute  alcohol  if  the  tissue  is  to  be 
embedded  at  once,  in  50  per  cent  spirit  if  it  is  to  be  kept  for  some  time.     For 
small  pieces  of  tissue  fixation  for  twelve  hours  or  even  less  is  sufficient.     The 
method  is  a  rapid  and  very  satisfactory  one. 

(c)  Corrosive  sublimate  is  an  excellent  fixative  agent.     It  is  best  used  as 
a  saturated  solution  in  .75  per  cent  sodium  chloride  solution.     Dissolve  the 
sublimate  in  the  salt  solution  by  heat ;  the  separation  of  crystals  on  cooling 
shows  that  the  solution  is  saturated.     For  small  pieces  of  tissue  \  inch  in 
thickness,  twelve   hours1  immersion   is   sufficient.     If  the  pieces  are  larger, 
twenty-four  hours  is  necessary.     They  should  then  be  tied  up  in  a  piece  of 
gauze,  and  placed  in  a  stream  of  running  water  for  from  twelve  to  twenty-four 
hours,  according  to  the  size  of  the  pieces,  to  wash  out  the  excess  of  sublimate. 
They  are  then  placed  for  twenty-four  hours  in  each  of  the  following  strengths 
of  methylated  spirit  (free  from  naphtha1)  :  30  per  cent,  60  per  cent,  and  90  per 
cent.     Finally  they  are  placed  in  absolute  alcohol  for  twenty-four  hours  and 
are  then  ready  to  be  prepared  for  cutting. 

If  the  tissue  is  very  small,  as  in  the  case  of  minute  pieces  removed  for 
diagnosis,  the  stages  may  be  all  compressed  into  twenty-four  hours.  In  fact, 
after  fixation  in  corrosive  the  tissue  may  be  transferred  directly  to  absolute 
alcohol,  the  perchloride  of  mercury  being  removed  after  the  sections  are  cut, 
as  will  be  afterwards  described. 

1  In  Britain  ordinary  commercial  methylated  spirit  has  wood  naphtha  added  to  it 
to  discourage  its  being  used  as  a  beverage.  The  naphtha  being  insoluble  in  water,  a 
milky  fluid  results  from  the  dilution  of  the  spirit.  By  law,  chemists  can  only  sell 
8  ounces  of  pure '  spirit  at  a  time.  Most  pathological  laboratories  are,  however, 
licensed  by  the  Excise  to  buy  pure  spirit  in  large  quantities. 


THE   CUTTING   OF    SECTIONS.  93 

(//)  Methylated  Spirit.  —  Small  pieces  of  tissue  may  be  placed  in  methy- 
lated spirit,  which  is  to  be  changed  after  the  first  day.  In  six  to  seven  days 
they  will  be  hardened.  If  the  pieces  are  large,  a  longer  time  is  necessary. 

The  Cutting  of  Sections.  —  i.  By  Means  of  tJte  Freezing 
Microtome.  —  Pieces  of  tissue  hardened  by  any  of  the  above 
methods  must  have  all  the  alcohol  removed  from  them  by  wash- 
ing in  running  water  for  twenty-four  hours.  They  are  then 
placed  for  from  twelve  to  twenty-four  hours  (according  to  their 
size)  in  a  thick  syrupy  solution  containing  two  parts  of  gum 
arabic  and  one  part  of  sugar.  They  are  then  cut  on  a  freezing 
microtome  (of  which  Cathcart's  is  a  good  example)  and  placed 
for  a  few  hours  in  a  bowl  of  water  so  that  the  gum  and  syrup 
may  dissolve  out.  They  are  then  stained,  or  they  may  be  stored 
in  methylated  spirit. 

2.  Embedding  and  Cutting  in  Solid  Paraffin.  —  This  method 
gives  by  far  the  finest  results,  and  should  always  be  adopted 
when  practicable.  The  principle  is  the  impregnation  of  the 
tissue  with  paraffin  in  the  melted  state.  This  paraffin  when  it 
solidifies  gives  support  to  all  the  tissue  elements.  The  method 
involves  that,  after  hardening,  the  tissue  shall  be  thoroughly 
dehydrated,  and  then  thoroughly  permeated  by  some  solvent 
of  paraffin  which  will  expel  the  dehydrating  fluid  and  prepare 
for  the  entrance  of  the  paraffin.  The  solvents  most  in  use  are 
chloroform,  cedar  oil,  xylol,  and  turpentine;  of  these  chloroform 
and  cedar  oil  are  the  best,  the  former  being  preferred  as  it  per- 
meates the  tissue  more  rapidly.  The  more  gradually  the  tissues 
are  changed  from  reagent  to  reagent  in  the  processes  to  be  gone 
through,  the  more  successful  is  the  result.  A  necessity  of  the 
process  is  an  oven  with  hot-water  jacket,  in  which  the  paraffin 
can  be  kept  at  a  constant  temperature  just  above  its  melting- 
point,  a  gas  regulator,  e.g.  Reichert's,  being  of  course  necessary. 
The  tissues  occurring  in  pathological  work  have  a  tendency  to 
become  brittle  if  overheated,  and  therefore  the  best  results  are 
not  obtained  by  using  paraffin  melting  about  58°  C,  such  as  is 
employed  in  most  biological  laboratories.  We  have  used  for 
some  years  a  mixture  of  one  part  of  paraffin  melting  at  48°  and 
two  parts  of  paraffin  melting  at  54°  C.  This  mixture  has  a 
melting-point  between  52°  and  53°  C.,  and  it  serves  all  ordinary 
purposes  well.  An  excellent  quality  of  paraffin  is  that  known 
as  the  "  Cambridge  paraffin,"  but  many  scientific-instrument 


94  MICROSCOPIC   METHODS. 

makers  supply  paraffins  which,  for  ordinary  purposes,  are  quite 
as  good,  and  much  cheaper.  The  successive  steps  in  the  pro- 
cess of  paraffin  embedding  are  as  follows  :  — • 

1.  Pieces  of  tissue,  however  hardened,  are  placed  in  fresh  absolute  alcohol 
for  twenty-four  hours  in  order  to  complete  their  dehydration. 

2.  Transfer  now  to  a  mixture  of  equal  parts  of  absolute  alcohol  and 
chloroform  for  twenty-four  hours. 

3.  Transfer  to  pure  chloroform  for  twenty-four  hours.     At  the  end  of 
this  time  the  tissues  should  sink,  or  float  heavily. 

4.  Transfer  now  to  a  mixture  of  equal  parts  of  chloroform  and  paraffin, 
and  place  on  the  top  of  the  oven  for  from  twelve  to  twenty-four  hours.     If 
the  temperature  there  is  not  sufficient  to  keep  the  mixture  melted,  then  they 
must  be  put  inside. 

5.  Place  in  pure  melted  paraffin  in  the  oven  for  twenty-four  hours.     For 
holding  the  paraffin  containing  the  tissues,  small  tin  dishes  such  as  are  used 
by  pastry-cooks  will  be  found  very  suitable.     There  must  be  a  considerable 
excess  of  paraffin  over  the  bulk  of  tissue  present,  otherwise  sufficient  chloro- 
form will  be  present  to  vitiate  the  final  result  and  not  give  the  perfectly  hard 
block  obtained  with  pure  paraffin.     With  experience,  the  persistence  of  the 
slightest  trace  of  chloroform  can  be  recognised  by  smell. 

In  the  case  of  very  small  pieces  of  tissue  the  time  given  for  each  stage 
may  be  much  shortened,  and  where  haste  is  desirable  Nos.  2  and  4  may  be 
omitted.  Otherwise  it  is  better  to  carry  out  the  process  as  described. 

6.  Cast  the  tissues  in  blocks  of  paraffin  as  follows :  Pairs  of  L-shaped 
pieces  of  metal  made  for  the  purpose  by  instrument  makers  must  be  at  hand. 
By  laying  two  of  these  together  on  a  glass  plate,  a  rectangular  trough  is  formed. 
This  is  filled  with  melted  paraffin  taken  from  a  stock  in  a  separate  dish.     In 
it  is  immersed  the  piece  of  tissue,  which  is  lifted  out  of  its  pure  paraffin  bath 
with  heate'd  forceps.     The  direction  in  which  it  is  to  be  cut  must  be  noted 
before  the  paraffin  becomes  opaque.     When  the  paraffin  has  begun  to  set, 
the  glass  plate  and  trough  have  cold  water  run  over  them.     When  the  block 
is  cold,  the  metal  Us  are  broken  off,  and,  its  edges  having  been  pared,  it  is 
stored  in  a  pill-box. 

The  Cutting  of  Paraffin  Sections.  —  Sections  must  be  cut  as 
thin  as  possible,  the  Cambridge  rocking  microtome  being,  on 
the  whole,  most  suitable.     They  should  not  exceed  8  UL  in  thick- 
ness, and  ought,  if  possible,  to  be  about  4  p.     For  their  manipu- 
lation it  is  best  to  have 
two  needles  on  handles, 
two  camel's-hair  brushes 

FlG.  51.  —  Needle  with  square  of  paper  on  end  for  in  i  n 

manipulating  paraffin  sections.  on  handles,  and  a  needle 

with  a  rectangle  of  stiff 

writing  paper  fixed  on  it  as  in  the  diagram  (Fig.  51).  When  cut, 
sections  are  floated  on  the  surface  of  a  beaker  of  water  kept  at  a 


PREPARATION   OF   SECTIONS   FOR   STAINING.  95 

temperature  about  10°  C.  below  the  melting-point  of  the  paraffin. 
On  the  surface  of  the  warm  water  they  become  perfectly  flat. 

Fixation  on  Ordinary  Slides.  (#)  Gulland"*s  Method.  — A  supply  of  slides 
well  cleaned  being  at  hand,  one  of  them  is  thrust  obliquely  into  the  water 
below  the  section,  a  corner  of  the  section  is  fixed  on  it  with  a  needle,  and  the 
slide  withdrawn.  The  surplus  of  water  being  wiped  off  with  a  cloth,  the  slide 
is  placed  on  a  support,  with  the  section  downwards,  and  allowed  to  remain  on 
the  top  of  the  paraffin  oven  or  in  a  bacteriological  incubator  for  from  twelve 
to  twenty-four  hours.  It  will  then  be  sufficiently  fixed  on  the  slide  to  with- 
stand all  the  manipulations  necessary  during  staining  and  mounting. 

(ti)  Fixation  by  Mann's  Method.  —  This  has  the  advantage  of  being  more 
rapid  than  the  previous  one.  A  solution  of  albumin  is  prepared  by  mixing 
the  white  of  a  fresh  egg  with  ten  parts  of  distilled  water  and  filtering.  Slides 
are  made  perfectly  clean  with  alcohol.  One  is  dipped  into  the  solution  and 
its  edge  is  then  drawn  over  one  surface  of  another  slide  so  as  to  leave  on  it 
a  thin  film  of  albumin.  This  is  repeated  with  the  others.  As  each  is  thus 
coated,  it  is  leant,  with  the  film  downwards,  on  a  ledge  till  dry,  and  then  the 
slides  are  stored  in  a  wide  stoppered  jar  till  needed.  The  floating  out  is 
performed  as  before.  The  albuminised  side  of  the  slide  is  easily  recognised 
by  the  fact  that  if  it  is  breathed  on,  the  breath  does  not  condense  on  it. 
The  great  advantage  of  this  method  is  that  the  section  is  fixed  after  twenty 
to  thirty  minutes'  drying  at  37°  C.  If  the  tissue  has  been  hardened  in  any 
of  the  bichromate  solutions  and  embedded  in  paraffin,  this  or  some  corre- 
sponding method  of  fixing  the  sections  on  the  slide  must  be  used. 

Preparation  of  Paraffin  Sections  for  Staining.  —  Before  stain- 
ing, the  paraffin  must  be  removed  from  the  section.  This  is 
best  done  by  dropping  on  xylol  out  of  a  drop-bottle.  When  the 
paraffin  is  dissolved  out,  the  superfluous  xylol  is  wiped  off  with 
a  cloth  and  a  little  absolute  alcohol  dropped  on.  When  the 
xylol  is  removed  the  superfluous  alcohol  is  wiped  off  and  a 
little  50  per  cent  methylated  spirit  dropped  on.  During  these 
procedures  sections  must  on  no  account  be  allowed  to  dry. 
The  sections  are  now  ready  to  be  stained.  Deposits  of  crystals 
of  corrosive  sublimate  often  occur  in  sections  which  have  been 
fixed  by  this  reagent.  These  can  be  readily  removed  by  placing 
the  sections,  before  staining,  for  a  few  minutes  in  equal  parts  of 
Gram's  iodine  solution  (p.  102)  and  water,  and  then  washing  out 
the  iodine  with  methylated  spirit. 

To  save  repetition  we  shall  in  treating  of  stains  suppose  that, 
with  paraffin  sections,  the  above  preliminary  steps  have  already 
been  taken,  and  further,  that  sections  cut  by  a  freezing  micro- 
tome are  also  in  spirit  and  water. 


96  MICROSCOPIC   METHODS. 

Dehydration  and  Clearing.  —  It  is  convenient,  first  of  all,  to 
indicate  the  final  steps  to  be  taken  after  a  specimen  is  stained. 
Dry  films  after  being  stained  are  washed  in  water,  dried  and 
mounted  in  xylol-balsam ;  wet  films  and  sections  must  be  dehy- 
drated, cleared,  and  then  mounted  in  xylol-balsam. 

Dehydration  is  most  commonly  effected  with  absolute  alco- 
hol. Alcohol,  however,  sometimes  decolorises  the  stained  or- 
ganisms more  than  is  desirable,  and  therefore  Weigert  devised 
the  following  method  for  dehydrating  and  clearing  by  aniline 
oil,  which,  though  it  may  decolorise  somewhat,  does  not  do  so 
to  the  same  extent  as  alcohol.  As  much  as  possible  of  the 
water  being  removed,  the  section  placed  on  a  slide  is  partially 
dried  by  draining  with  fine  blotting  paper.  Some  aniline  oil  is 
placed  on  the  section  and  the  slide  moved  to  and  fro.  The  sec- 
tion is  dehydrated  and  becomes  clear.  The  process  may  be 
accelerated  by  heating  gently.  The  preparation  is  then  treated 
with  a  mixture  of  two  parts  of  aniline  oil  and  one  part  of  xylol, 
and  then  with  xylol  alone,  after  which  it  is  mounted  in  xylol- 
balsam.  Paraffin  sections  can  usually  be  dehydrated  and  cleared 
by  the  mixture  of  aniline  oil  and  xylol  alone. 

Sections  stained  for  bacteria  should  always  be  cleared,  at 
least  finally,  in  xylol,  for  the  same  reason  that  xylol-balsam  is 
to  be  used  for  mounting  films,  viz.,  that  it  dissolves  out  aniline 
dyes  less  readily  than  such  clearing  reagents  as  clove  oil,  etc. 
Xylol,  however,  requires  the  previous  dehydration  to  have  been 
more  complete  than  clove  oil,  which  will  clear  a  section  readily 
when  the  dehydration  has  been  only  partially  effected  by,  say, 
methylated  spirit.  If  a  little  decolorisation  of  a  section  is  still 
required  before  mounting,  clove  oil  may  be  used  to  commence 
the  clearing,  the  process  being  finished  with  xylol.  With  a  little 
experience  the  progress,  not  only  of  these  processes  but  also 
of  staining,  can  be  very  accurately  judged  of  by  observing  the 
appearance  under  a  low  objective. 

THE  STAINING  OF  BACTERIA. 

Staining  Principles.  —  To  speak  generally,  the  protoplasm  of 
bacteria  reacts  to  stains  in  a  manner  similar  to  the  nuclear 
chromatin,  though  sometimes  more  and  sometimes  less  actively. 
The  bacterial  stains  par  excellence  are  the  basic  aniline  dyes. 


THE   STAINING   OF   BACTERIA. 


97 


These  dyes  are  more  or  less  complicated  compounds  derived 
from  the  coal-tar  product  aniline  (C6H5  .  NH2).  Many  of  them 
have  the  constitution  of  salts.  Such  compounds  are  divided 
into  two  groups  according  as  the  staining  action  depends  on  the 
basic  or  the  acid  portion  of  the  molecule.  Thus  the  acetate  of 
rosaniline  derives  its  staining  action  from  the  rosaniline.  It 
is  therefore  called  a  basic  aniline  dye.  On  the  other  hand, 
ammonium  picrate  owes  its  action  to  the  picric  acid  part  of  the 
molecule.  It  is  therefore  termed  an  acid  aniline  dye.  These 
two  groups  have  affinities  for  different  parts  of  the  animal  cell. 
The  basic  stains  have  a  special  affinity  for  the  nuclear  chromatin, 
the  acid  for  the  protoplasm  and  various  formed  elements.  Thus 
it  is  that  the  former  —  the  basic  aniline  dyes  —  are  especially 
the  bacterial  stains. 

The  number  of  basic  aniline  stains  is  very  large.  The  following  are  the 
most  commonly  used  : 1  — 

Violet  Stains.  —  Methyl-violet,    R-$R    (synonyms :    Hoffmann's    violet, 
dahlia). 

Gentian-violet  (synonyms:  benzyl-violet,  Pyoktanin). 

Crystal  violet. 
Blue  Stains. — Methylene-blue 2  (synonym:  phenylene-blue). 

Victoria-blue. 

Thionin-blue. 
Red  Stains.  —  Basic  fuchsin  (synonyms:  basic  rubin,  magenta). 

Safranin  (synonyms:  fuchsia,  Girofle). 

Brown   Stain.  —  Bismarck-brown      (synonyms :      vesuvin,     phenylene- 
brown). 

It  is  of  the  greatest  importance  that  the  stains  used  by  the 
bacteriologist  should  be  good,  and  therefore  it  is  advisable  to 
obtain  those  prepared  by  Griibler  of  Leipzig.  One  is  then  per- 
fectly sure  that  one  has  got  the  right  stain. 

Of  the  stains  specified,  the  violets  and  reds  are  the  most 
intense  in  action,  especially  the  former.  It  is  thus  easy  in  using 
them  to  overstain  a  specimen.  Of  the  blues,  methylene-blue 
probably  gives  the  best  differentiation  of  structure,  and  it  is 
difficult  to  overstain  with  it.  Thionin-blue  also  gives  good  dif- 
ferentiation and  does  not  readily  overstain.  Its  tone  is  deeper 

1  For   further  information  on  this  subject   the  student  is  referred  to  Rawitz, 
"Leitfaden  fur  histologische  Untersuchungen,"  Jena,  1895,  from  which  the  synonyms 
used  in  the  text  are  taken. 

2  This  is  to  be  distinguished  from  methyl-blue,  which  is  a  different  compound. 


98 


MICROSCOPIC   METHODS. 


than  that  of  methylene-blue  and  it  approaches  the  violets  in  tint. 
Bismarck-brown  is  a  weak  stain,  but  is  useful  for  some  purposes. 
Formerly  it  was  much  used  in  photomicrographic  work,  as  it 
was  less  actinic  than  the  other  stains.  It  is  not,  however, 
needed  now,  on  account  of  the  improved  sensitiveness  of  plates. 
It  is  most  convenient  to  keep  saturated  alcoholic  solutions  of 
the  stains  made  up,  and  for  use  to  filter  a  little  into  about  ten 
times  its  bulk  of  distilled  water  in  a  watch-glass.  A  solution  of 
good  body  is  thus  obtained.  Most  bacteria  (except  those  of 
tubercle,  leprosy,  and  a  few  others)  will  stain  in  a  short  time  in 
such  a  fluid.  Watery  solutions  may  also  be  made  up,  e.g.  a 
saturated  watery  solution  of  methylene-blue  or  a  i  per  cent 
solution  of  gentian-violet.  Stains  must  always  be  filtered  before 
use ;  otherwise  there  may  be  deposited  on  the  preparation 
granules  which  it  is  impossible  to  wash  off.  The  violet  stains 
in  solution  in  water  have  a  great  tendency  to  decompose.  Only 
small  quantities  should  therefore  be  prepared  at  a  time. 

The  Staining  of  Cover-glass  Films.  —  Films  are  made  from 
cultures  as  described  above.  The  cover-glass  may  be  floated  on 
the  surface  of  the  stain  in  a  watch-glass, 
or  the  cover-glass,  held  in  forceps  with  the 
film  side  uppermost,  may  have  as  much 
stain  poured  on  it  as  it  will  hold.  When 
the  preparation  has  been  exposed  for  the 
requisite  time,  usually  a  few  minutes,  it  is 
well  washed  in  tap  water  in  a  bowl,  or  with 
distilled  water  with  such  a  simple  contriv- 
ance as  that  figured  (Fig.  52).  The  figure 
explains  itself.  When  the  film  has  been 
washed,  the  surplus  of  water  is  drawn  off 
with  a  piece  of  filter  paper,  the  preparation 
is  carefully  dried  high  over  a  flame,  a  drop 
of  xylol-balsam  is  applied,  and  the  cover- 
glass  mounted  on  a  slide.  It  is  sometimes 
advantageous  to  examine  films  in  a  drop  of 
water  in  place  of  balsam.  The  films  can 
be  subsequently  dried  and  mounted  perma- 
nently. In  the  case  of  tubercle,  special 


FIG.  52.  —  Syphon  wash- 
bottle  for  distilled  water  used 
in  washing  preparations. 


stains  are  necessary  (p.  104),  but  with  this  exception,  practically 
all  bacterial  films  made  from  cultures  can  be  stained  in  this  way. 


USE   OF   MORDANTS   AND   DECOLORISERS.  99 

Some  bacteria,  e.g.  typhoid,  glanders,  take  up  the  stains  rather 
slowly,  and  for  these  the  more  intensive  stains,  red  or  violet, 
are  to  be  preferred. 

Films  of  fluids  from  the  body  (blood,  pus,  etc.)  can  be  gen- 
erally stained  in  the  same  way,  and  this  is  often  quite  sufficient 
for  diagnostic  purposes.  The  blue  dyes  are  here  preferable,  as 
they  do  not  readily  overstain.  Should  overstaining  occur  it  is 
easily  remedied  by  decolorising  for  a  few  seconds  in  glacial  acetic 
acid,  i-iooo,  and  removing  the  acid  by  thoroughly  washing  in 
water.  In  the  case  of  such  fluids,  if  the  histological  elements 
also  claim  attention,  it  is  best  first  to  stain  the  cellular  proto- 
plasm with  a  one  to  two  per  cent  watery  solution  of  eosin  (which 
is  an  acid  dye),  and  then  to  use  a  blue  which  will  stain  the 
bacteria  and  the  nuclei  of  the  cells.  In  the  case  of  films  made 
from  urine,  where  there  is  little  or  no  albuminous  matter  present, 
the  bacteria  may  be  imperfectly  fixed  on  the  slide,  and  are  thus 
apt  to  be  washed  off.  In  such  a  case  it  is  well  to  modify  the 
staining  method.  A  drop  of  stain  is  placed  on  a  slide,  and 
the  cover-glass,  film  side  down,  lowered  upon  it.  After  the 
lapse  -of  the  time  necessary  for  staining,  a  drop  of  water  is 
placed  at  one  side  of  the  cover-glass  and  a  little  piece  of  filter 
paper  at  the  other  side.  The  result  is  that  the  stain  is  sucked  out 
by  the  filter  paper.  By  adding  fresh  drops  of  water  and  using 
fresh  pieces  of  filter  paper,  the  specimen  is  washed  without  any 
violent  application  of  water,  and  the  bacteria  are  not  displaced. 

For  the  general  staining  of  films  a  saturated  watery  solution  of 
methylene-blue  will  be  found  to  be  the  best  stain  to  commence 
with. 

The  Use  of  Mordants  and  Decolorising  Agents.  —  In  films  of 
blood  and  pus,  and  still  more  so  in  sections  of  tissues,  if  the  above 
methods  are  used,  the  tissue  elements  may  be  stained  to  such  an 
extent  as  to  quite  obscure  the  bacteria.  Hence  many  methods 
have  been  devised  in  which  the  general  principle  may  be  said  to 
be  (a)  the  use  of  substances  which,  while  increasing  the  staining 
power,  tend  to  fix  the  stain  in  the  bacteria,  and  (#)  the  subsequent 
treatment  by  substances  which  decolorise  the  overstained  tissues 
to  a  greater  or  less  extent,  while  they  leave  the  bacteria  coloured. 
The  staining  capacity  of  a  solution  may  be  increased  — 

(a)  By  the  addition  of  substances  such  as  carbolic  acid,  aniline 
oil,  or  metallic  salts,  all  of  which  probably  act  as  mordants. 


100  MICROSCOPIC  METHODS. 

(fr)  By  the  addition  of  alkalies,  such  as  caustic  potash  or 
ammonium  carbonate,  in  weak  solution. 

(c)   By  the  employment  of  heat. 

{d}  By  long  duration  of  the  staining  process. 

As  decolorisjng  agents  we  use  chiefly  mineral  acids  (hydro- 
chloric, nitric,  sulphuric),  vegetable  acids  (especially  acetic  acid), 
alcohol  (either  methylated  spirit  or  absolute  alcohol),  or  a  com- 
bination of  spirit  and  acid,  e.g.  methylated  spirit  with  a  drop  or 
two  of  hydrochloric  acid  added,  also  various  oils,  e.g.  aniline, 
clove,  etc.  In  most  cases  about  thirty  drops  of  acetic  acid  in  a 
bowl  of  water  will  be  sufficient  to  remove  the  excess  of  stain  from 
overstained  films  and  sections.  More  of  the  acid  may,  of  course, 
be  added  if  necessary. 

Hot  water  also  decolorises  to  a  certain  extent ;  overstained 
films  can  be  readily  decolorised  by  placing  a  drop  of  water  on 
the  film  and  heating  gently  over  a  flame. 

When  preparations  have  been  sufficiently  decolorised  by  an 
acid,  they  should  be  well  washed  in  tap  water,  or  in  distilled 
water  with  a  little  lithia  carbonate  added. 

The  methods  embracing  the  use  of  a  stain  with  a  mordant, 
and  a  decoloriser,  are  very  numerous,  and  we  can  only  enumerate 
the  best  of  them. 

We  sometimes  have  to  deal  with  bacteria  which  show  a 
special  tendency  to  be  decolorised.  This  tendency  can  be 
obviated  by  adding  a  little  of  the  stain  to  the  alcohol,  or  aniline 
oil,  employed  in  dehydration.  In  the  latter  case  a  little  of  the 
stain  is  rubbed  down  in  the  oil.  The  mixture  is  allowed  to 
stand.  After  a  little  time  a  clear  layer  forms  on  the  top  with 
stain  in  solution,  and  this  can  be  drawn  off  with  a  pipette. 

When  methylene-blue,  methyl-violet,  or  gentian-violet  is  used 
the  stain  can,  after  the  proper  degree  of  decolorisation  has 
been  reached,  be  fixed  in  the  tissues  by  treating  for  a  minute 
with  ammonium  molybdate  (2*  per  cent  in 'water). 

The  Formulae  of  some  of  the  more  commonly  used  Stain  Combinations, 
i .   L'offler*s  Methylene-blue. 

Saturated  solution  of  methylene-blue  in  alcohol    .         .         .         .30  c.c. 
Solution  of  potassium  hydrate  in  distilled  water  (1-10,000)  .         .         100  c.c. 

(This  dilute  solution  may  be  conveniently  made  by  adding  I  c.c.  of  a 
i  per  cent  solution  to  99  c.c.  of  water.) 


FORMULA   OF  %£ 

Sections  may  be  stained  in  this  mixture  for  from  a  quarter  of  an  hour  to 
several  hours.  They  do  not  readily  overstain.  The  tissue  containing  the 
bacteria  is  then  decolorised  if  necessary  with  |-i  per  cent  acetic  acid,  till  it  is 
a  pale  blue-green.  The  section  is  washed  in  water,  rapidly  dehydrated  with 
alcohol  or  aniline  oil,  cleared  in  xylol,  and  mounted. 

The  tissue  may  be  contrast  stained  with  eosin.  If  this  is  desired,  after 
decolorisation  wash  with  water,  place  for  a  few  seconds  in  i  per  cent  solution 
of  eosin  in  absolute  alcohol,  rapidly  complete  dehydration  with  pure  absolute 
alcohol,  and  proceed  as  before. 

Films  may  be  stained  with  Loffler's  blue  by  five  minutes1  exposure  or 
longer  in  the  cold.  They  usually  do  not  require  decolorisation,  as  the  tissue 
elements  are  not  overstained. 

2.  Kuhne^s  Methylene-blue. 

Methylene-blue          .         .         .         .  i-5  gr. 

Absolute  alcohol        .         .  .  10  c.c. 

Carbolic  acid  solution  (1-20)     .         .         100    „ 

Stain  and  decolorise  as  with  LofHer's  blue,  or  decolorise  with  very  weak 
hydrochloric  acid  (a  few  drops  in  a  bowl  of  water) . 

3.  Carbol-thionin-blue. — Make  up  a  stock  solution  consisting  of  i  gramme 
of  thionin-blue  dissolved  in  100  c.c.  carbolic  acid  solution  (1-40).     For  use, 
dilute  i  volume  with  3  of  water  and  filter.     Stain  sections  for  five  minutes  or 
upwards.     Wash  very  thoroughly  With  water,  otherwise  a  deposit  of  crystals 
may  occur  in  the  subsequent  stages.     Decolorise  with  very  weak  acetic  acid. 
A  few  drops  of  the  acid  added  to  a  bowl  of  water  is  quite  sufficient.     Wash 
again  thoroughly  with  water.     Dehydrate  with   absolute  alcohol.     Thionin- 
blue  stains  more  deeply  than  methylene-blue,  and  gives  equally  good  differ- 
entiation.    It  is  very  suitable  for  staining  typhoid  and   glanders  bacilli   in 
sections.     Cover-glass  preparations  stained  by  this  method   do  not  usually 
require  decolorisation.     As  a  contrast  stain,  i  per  cent  watery  solution  of 
eosin  may  be  used  before  staining  with  the  thionin. 

4.  Gentian-violet  in  Aniline  Oil  Water.  —  Two  solutions  have  here  to  be 
made  up.     (a)  Aniline  oil  water.     Add  exactly  2  c.c.  aniline  oil  to  98  c.c. 
distilled  water  in  a  flask,  and  shake  violently  till  as  much  as  possible  of  the 
oil  has  dissolved.     Filter  twice  through  the  same  paper  and  keep  in  a  covered 
bottle  to  prevent  access  of  light,     (b}  Make  a  saturated  solution  of  gentian- 
violet  in  alcohol.     When  the  stain  is  to  be  used,  25  parts  of  (6)  is  added  to 
75  parts  of  (#),  and  the  mixture  filtered.     This  mixture  will  not  readily  de- 
compose and  may  be  used  for  several  months  if  kept  from  the  prolonged  action 
of  light,  although  it  may  require  occasional  filtering  to  remove  accumulated 
crystalline  deposit.     Stain  sections  for  a  few  minutes ;  then  decolorise  with 
methylated  spirit.     Sometimes  it  is  advantageous  to  add  to  the  methylated 
spirit  a  little   hydrochloric  acid  (2-3   minims   to   100  c.c.).     This   staining 
solution  is  not  so  much  used  by  itself  for  tissues,  as  in  Gram's  method,  which 
is  presently  to  be  described,  but  makes  an  excellent  stain  for  most  bacterial 
films. 

5.  Carbol-gentian-violet.  —  i  part  saturated  alcoholic  solution  of  gentian- 


j  ^  >;{^;  i       MICROSCOPIC  METHODS. 

violet  is  mixed  with  10  parts  of  5  per  cent  solution  of  carbolic  acid.     It  is 
used  as  No.  4. 

6.  Carbol-fuchsin  (see  p.  104).  —  This  is  a  very  powerful  stain,  and,  when 
used  in  the  undiluted  condition,  £-1  minute's  staining  is  usually  sufficient.  It 
is  better,  however,  to  dilute  with  five  to  ten  times  its  volume  of  water  and 
stain  for  a  few  minutes.  In  this  form  it  has  a  very  wide  application. 
Methylated  spirit  with  or  without  a  few  drops  of  acetic  acid  is  the  most  con- 
venient decolorising  agent.  Then  dehydrate  thoroughly,  clear,  and  mount. 

Various  other  staining  combinations  might  be  given,  but  the 
above  are  the  best  and  most  widely  used.  If  the  reader  has 
thoroughly  grasped  the  remarks  made  above  on  the  general 
principles  which  underlie  the  staining  of  bacteria,  he  will  be  able 
to  use  any  combination  to  which  his  attention  may  be  directed. 
We  may  only  add  here  that  different  organisms  take  up  and  hold 
different  stains  with  different  degrees  of  intensity,  and  thus 
duration  of  staining  and  degree  of  decolorisation  must  be  varied. 
It  may  be  laid  down  as  a  general  rule  that,  so  long  as  organisms 
retain  the  stain,  the  greater  the  decolorisation  of  the  tissues  in 
which  they  lie,  the  clearer  will  be  the  results. 

Gram's  Method  and  its  Modifications.  —  In  the  methods 
already  described  the  tissues,  and  more  especially  the  nuclei, 
retain  some  stain  when  decolorisation  has  reached  the  point  to 
which  it  can  safely  go  without  the  bacteria  themselves  being 
affected.  In  the  method  of  Gram,  now  to  be  detailed,  this  does 
not  occur,  for  the  stain  can  here  be  removed  completely  from  the 
ordinary  tissues,  and  left  only  in  the  bacteria.  All  kinds  of 
bacteria,  however,  do  not  retain  the  stain  in  this  method,  and 
'therefore  in  the  systematic  description  of  any  species  it  is 
customary  to  state  whether  it  is,  or  is  not,  stained  by  Gram's 
method  —  by  this  is  meant,  as  will  be  understood  from  what  has 
been  said,  whether  the  particular  organism  retains  the  colour  after 
the  latter  has  been  completely  removed  from  the  tissues.  It  must, 
however,  be  remarked  that  some  tissue  elements  may  retain  the 
stain  as  firmly  as  any  bacteria,  e.g.  keratinised  epithelium,  calci- 
fied particles,  the  granules  of  mast  cells,  and  sometimes  altered 
red  blood  corpuscles,  etc. 

In  Gram's  method  the  essential  feature  is  the  treating  of  the 
tissue,  after  staining,  with  a  solution  of  iodine.  This  solution  is 
spoken  of  as  Gram's  solution  (sometimes  as  Lugol's),  and  has 
the  following  composition  :  — 


GRAM'S    STAIN.  103 

Iodine i  part. 

Potassium  iodide         ....  2  parts. 

Distilled  water    .         .         .         .         .         300     „ 

The  following  is  the  method  :  — 

1.  Stain  in  aniline   oil  gentian-violet  or   in   carbol-gentian-violet  (vide 
pp.  101,  102)  for  about  five  minutes,  and  wash  in  water. 

2.  Treat  the  section  or  film  with  Gram's  solution  till  its  colour  becomes  a 
purplish  black  —  generally  about  half  a  minute  or  a  minute  is  sufficient  for  the 
action  to  take  place.  \ 

3.  Decolorise  with  absolute  alcohol  or  methylated  spirit  till  t{ie  colour  has 
almost  entirely  disappeared,  the  tissues  having  only  a  faint  violet  tint. 

4.  Dehydrate  completely,  clear  with  xylol,  and  mount.     In  the  case  of  film 
preparations,  the  specimen  is  simply  washed  in  water,  dried,  and  mounted. 

In  stage  (3)  the  process  of  decolorisation  is  more  satisfactorily  performed 
by  using  clove  oil  after  sufficient  dehydration  with  alcohol,  the  clove  oil  being 
afterwards  removed  by  xylol. 

As  a  contrast  stain  for  the  tissues  carmalum  or  lithia  carmine  is  used  before 
staining  with  gentian-violet  (i).  As  a  contrast  stain  for  other  bacteria  which 
are  decolorised  by  Gram's  method  carbol-fuchsin  diluted  with  ten  volumes  of 
water,  or  a  saturated  watery  solution  of  Bismarck-brown,  may  be  used  before 
stage  (4). 

As  applied  to  bacterial  films  special  conditions  must  be 
observed.  The  film  must  always  be  prepared  from  an  agar 
slant-culture  between  12  and  24  hours  old,  and  is  then  to  be 
treated  as  follows  :  — 

1.  Stain  in  aniline  gentian-violet  for  i|  minutes. 

2.  Wash  in  water. 

3.  Stain  in  Gram's  solution  for  \\  minutes. 

4.  Decolorise  in  absolute  alcohol  for  at  least  four  minutes,  or  until  all 
stain  is  completely  discharged. 

5.  Mount  in  balsam. 

The  following  modifications  of  Gram's  method  may  be 
given :  — 

1.  Weigerfs  Modification. — The   contrast  staining   of  the   tissues   and 
stages   (i)  and  (2)  are  performed  as  above. 

(3)  After  using  the  iodine  solution  the  preparation  is  dried  by  blotting 
and  then  decolorised  by  aniline-xylol  (aniline  oil  2,  xylol  i). 

(4)  Wash  well  in  xylol  and  mount  in  xylol-balsam.     Film  preparations 
after  being  washed  in  xylol  may  be  dried,  and  thereafter  dilute  carbol-fuchsin 
may  be  used  to  stain  bacteria  which  have  been  decolorised. 

This  modification  probably  gives  the  most  uniformly  successful  results. 

2.  Nicolle*s  Modification.  —  Carbol-gentian-violet   is   used  as  the  stain. 
Treatment  with  iodine  is  carried  out  as  above  and  decolorisation  is  effected 
with  a  mixture  of  acetone  (i  part)  and  alcohol  (2  parts). 


104  MICROSCOPIC   METHODS. 

3.  Kuhnfs  Modification. — (i )  Stain  for  five  minutes  in  a  solution  made  up 
of  equal  parts  of  saturated  alcoholic  solution  of  crystal-violet  ("Krystall-violet") 
and  i  per  cent  solution  of  ammonium  carbonate. 

(2)  Wash  in  water. 

(3)  Place  for  two  to  three  minutes  in  Gram's  iodine  solution,  or  in  the 
following  modification  by  Kiihne  : — 

M 

Iodine 2  parts. 

Potassium  iodide        .         .         .         .  4      „ 

Distilled  water 100      „ 

For  use,  dilute  with  water  to  make  a  sherry-coloured  solution. 

(4)  Wash  in  water. 

(5)  Decolorise  in  a  saturated  alcoholic  solution  of  fluorescein  (a  saturated 
solution  in  methylated  spirit  does  equally  well). 

(6)  Dehydrate,  clear,  and  mount. 

Stain  for  Tubercle  and  Other  Acid-fast  Bacilli. — These  bacilli 
cannot  be  well  stained  with  a  simple  watery  solution  of  a  basic 
aniline  dye.  This  fact  can  easily  be  tested  by  attempting  to 
stain  a  film  of  a  tubercle  culture  with  such  a  solution.  They 
require  a*  powerful  stain  containing  a  mordant,  and  must  be 
exposed  to  the  stain  for  a  long  time,  or  the  action  of  the  latter 
may  be  aided  by  a  short  application  of  heat.  When  once 
stained,  however,  they  resist  decolorising  even  with  very  power- 
ful acids ;  they  are  therefore  called  "  acid-fast"  The  smegma 
bacillus  also  resists  decolorising  with  strong  acids  (p.  256),  and 
a  number  of  other  acid-fast  bacilli  have  recently  been  discovered 
(p.  254).  Any  combination  of  gentian-violet  or  fuchsin  with 
aniline  oil  or  carbolic  acid  or  other  mordant  will  stain  the 
bacilli  named,  but  the  following  methods  are  most  commonly 
used :  — 

Ziekl-Neelsen  Carbol-fuchsin  Stain. 

Basic  fuchsin i  part. 

Absolute  alcohol      .         .         .         .  10  parts. 

Solution  of  carbolic  acid  (1-20)        .         100     „ 

* 

1.  Place  the  specimen  in  this  fluid,  and  having  heated  it  till  steam  rises,. 
allow  it  to  remain  there  for  five  minutes,  or  allow  it  to  remain  in  the  cold 
stain  for  from  twelve  to  twenty-four  hours.      (Films  and  paraffin  sections  are 
usually  stained  with  hot  stain,  loose  sections  with  cold ;  in  hot  stain  the  latter 
shrink.) 

2.  Decolorise  with  20  per  cent  solution  of  strong  sulphuric  acid,  nitric 
acid,  or  hydrochloric  acid,  in  water.     In  this  the  tissues  become  yellow. 


STAIN   FOR  TUBERCLE   BACILLI.  IO5 

3.  Wash  well  with  water.     The  tissues  will  regain  a  faint  pink  tint.    If 
the  colour  is  distinctly  red,  the  decolorisation  is  insufficient,  and  the  specimen 
must  be  returned  to  the  acid.     As  a  matter  of  practice,  it  is  best  to  remove 
the  preparation  from  the  acid  every  few  seconds  and  wash  in  water,  replacing 
the  specimen  in  the  acid  and  re-washing  till  the  proper  pale  pink  tint  is  ob- 
tained.    Then  wash  in  alcohol  for  half  a  minute  and  replace  in  water. 

4.  Contrast  stain  with  a  saturated  watery  solution   of  methylene-blue 
for  half  a  minute,  or  with  saturated  Bismarck-brown  for  from  two  to  three 
minutes. 

5.  Wash  well  with  water.     In  the  case  of  films,  dry  and  mount.     In  the 
case  of  sections,  dehydrate,  clear,  and  mount. 

Fraenkel's  Modification  of  the  Ziehl-Neelsen  Stain. 

Here  the  process  is  shortened  by  using  a  mixture  containing 
both  the  decolorising  agent  and  the  contrast  stain. 

The  sections  or  films  are  stained  with  the  carbol-fuchsin  as  above  de- 
scribed, and  then  placed  in  the  following  decolorising  solution  :  — 

Distilled  water       .         .         .         .         .  50  parts e 

Absolute  alcohol    .         .         .         .         .  30    „ 

Nitric  acid  20     „ 

Methylene-blue  in  crystals  to  saturation. 

They  are  treated  with  this  till  the  red  colour  has  quite  disappeared  and  been 
replaced  by  blue.     The  subsequent  stages  are  the  same  as  in  No.  5,  siipra. 

Leprosy  bacilli  are  stained  in  the  same  way,  but  are  rather 
more  easily  decolorised  than  tubercle  bacilli,  and  it  is  better  to 
use  only  5  per  cent  sulphuric  acid  in  decolorising. 

In  the  case  of  specimens  stained  either  by  the  original  Ziehl- 
Neelsen  method,  or  by  Fraenkel's  modification,  the  tubercle  or 
leprosy  bacilli  ought  to  be  bright  red,  and  the  tissue  blue  or 
brown,  according  to  the  contrast  stain  used.  Other  bacteria 
which  may  be  present  are  also  coloured  with  the  contrast  stain. 

* 
Gabbetfs  Methylene-blue  Solution. 

Methylene-blue  .....         2  grms. 
Sulphuric  acid     .....       25  c.c. 
Distilled  water    .         ...         .       75  c.c. 

This  is  very  similar  to  Fraenkel's  decoloriser  and  contrast 
stain,  and  is  used  in  the  same  manner.  It  has  this  one  disad- 
vantage, that  it  will  not  decolorise  smegma  bacilli  should  these 
be  present  in  urinary  sediments  or  in  tissues  from  portions  of 
the  genito-urinary  tract. 


106  MICROSCOPIC   METHODS. 

The  Staining  of  Spores.  —  If  bacilli  containing  spores  are 
stained  with  a  watery  solution  of  a  basic  aniline  dye,  the  spores 
remain  unstained.  The  spores  either  take  up  the  stain  less 
readily  than  the  protoplasm  of  the  bacilli  or  they  have  a  resist- 
ing envelope  which  prevents  the  stain  penetrating  to  the  proto- 
plasm. Like  the  tubercle  bacilli,  when  once  stained  they  retain 
the  colour  with  considerable  tenacity.  The  following  is  the 
simplest  method  for  staining  spores :  - 

i.  Stain  cover-glass  films  as  for  tubercle  bacilli. 

2'.  Decolorise  with  i  per  cent  sulphuric  acid  in  water  or  with  methylated 

spirit.  This  removes  the  stain  from  the  bacilli. 

3.  Wash  in  water. 

4.  Stain  with  saturated  watery  methylene-blue  for  half  a  minute. 

5.  Wash  in  water,  dry,  and  mount  in  balsam. 

The  result  is  that  the  spores  are  stained  red,  the  protoplasm  of  the 
bacilli  blue. 

The  spores  of  some  organisms  lose  the  stain  more  readily  than  those  of 
others,  and  for  some,  methylated  spirit  is  a  sufficiently  strong  decolorising 
agent  for  use.  If  sulphuric  acid  stronger  than  i  per  cent  is  used,  the  spores  of 
many  bacilli  are  readily  decolorised. 

Moller*s  Method.  —  The  following  method,  recommended  by  Mb'ller,  is 
much  more  satisfactory  than  the  previous.  Before  being  stained,  the  films  are 
placed  in  chloroform  for  2  minutes,  and  then  in  a  5  per  cent  solution  of  chromic 
acid  for  $-2  minutes,  the  preparation  being  well  washed  after  each  reagent. 
Thereafter  they  are  stained  and  decolorised  as  above. 

The  Staining  of  Capsules.  —  The  two  following  methods  may 
be  recommended  in  the  case  of  capsulated  bacteria :  — 

(a}  Welches  Method.  —  This  depends  on  the  fact  that  in  many  cases  trie 
capsules  can  be  fixed  with  glacial  acetic  acid. 

Films  when  fixed  are  placed  in  this  acid  for  a  few  seconds. 

The  superfluous  acid  is  removed  with  filter  paper  and  the  preparation  is 
treated  with  gentian-violet  in  aniline-oil  water  repeatedly  till  all  the  acetic  acid 
is  removed. 

Then  wash  with  0.85-2.0  per  cent  solution  of  sodium  chloride  and  examine 
in  the  same  solution. 

Occasionally  such  preparations  can  be  kept  permanently  in  a  balsam 
mounting. 

The  capsule  appears  as  a  pale  violet  halo  around  the  deeply  stained 
bacterium. 

(b)  Richard  Muir^s  Method.  —  The  following  fixative  and  mordant  is 
made  up. 

Saturated  watery  solution  of  corrosive  sublimate  .  2 
Tannic  acid  solution  —  20  per  cent  ...  .  .2 
Saturated  solution  of  potash  alum  5 


THE   STAINING   OF   FLAGELLA.  *IO/ 

1 .  Films  containing  the  bacteria  are  dried  and  then  fixed  in  the  above  fluid 
for  two  minutes. 

2.  Wash  in  water,  then  in  spirit,  and  again  in  water. 

3.  Stain  with  carbol-fuchsin  for  2-3  minutes,  heating  gently. 

4.  Wash  in  water,  place  the  film  in  the  mordant  for  2-3  minutes,  and  wash 
again  in  water. 

5.  Stain    for   2    minutes  in   a  saturated  watery  solution    of  methylene- 
blue. 

6.  Differentiate  in  methylated  spirit,  dehydrate  in  alcohol,  clear  in  xylol, 
and  mount  in  xylol-balsam. 

The  bacteria  are  deep  crimson  and  the  capsules  of  a  blue  tint.  Fig.  79  is 
from  a  film  stained  by  this  method. 

The  Staining  of  Flagella.  —  The  staining  of  the  flagella  of 
bacteria  is  the  most  difficult  of  all  bacteriological  procedures, 
and  it  requires  considerable  practice  to  ensure  that  good  results 
shall  be  obtained.  Many  methods  have  been  introduced,  of 
which  the  three  following  are  the  most  satisfactory. 

Preparation  of  Films.  —  In  all  the  methods  of  staining  fla- 
gella, young  cultures  on  agar  should  be  used,  say  a  culture  in- 
cubated for  from  twelve  to  eighteen  hours  at  37°  C.  A  very 
small  portion  of  the  growth  is  taken  on  the  point  of  a  platinum 
needle  and  carefully  mixed  in  a  little  water  in  a  watch-glass  ;  the 
amount  should  be  such  as  to  produce  scarcely  any  turbidity  in 
the  water.  A  film  is  then  made  by  placing  a  drop  on  a  clean 
cover-glass  and  carefully  spreading  it  out  with  the  needle.  It  is 
allowed  to  dry  in  the  air,  and  is  then  passed  twice  or  thrice  through 
a  flame,  care  being  taken  not  to  overheat  it.  But  as  ordinarily 
practised,  there  is  far  too  much  handling  of  the  bacteria  in  the 
preparing  of  films,  whereby  large  numbers  of  the  organisms  are 
more  or  less  denuded  of  their  flagella,  in  consequence  giving 
poor  results.  To  avoid  this,  Kendall  recommends  the  following 
procedure  :  A  tube  containing  5  c.c.  of  sterile  water  is  gently  in- 
oculated with  enough  of  an  1 8-24-hour-old  agar  culture  of  a  bac- 
terium to  produce  a  very  faint  turbidity  in  the  upper  half  of  the 
water.  The  tube  is  then  placed  in  the  thermostat  for  one  hour, 
so  as  to  let  any  clumps  sediment  as  much  as  possible  and  per- 
mit of  slight  development.  Without  disturbing  the  fluid  in  any 
manner,  two  or  three  loopfuls  of  this  culture  are  placed  upon  a 
clean  cover-slip,  without  spreading,  and  dried  in  the  thermostat, 
when  they  are  to  be  fixed  in  the  flame  and  stained  by  any  of  the 
methods  recommended.  The  cover-glasses  used  should  always 


108  MICROSCOPIC   METHODS. 

be  cleaned  in  the  mixture  of  sulphuric  acid  and  potassium  bi- 
chromate described  on  page  89. 

/ 

i .   Pitfield  V  Method  as  modified  by  Richard  Muir. 

Prepare  the  following  solutions  :  — 

A.  The  Mordant. 

Tannic  acid,  10  per  cent  watery  solution,  filtered  .  10  c.c. 
Corrosive  sublimate,  saturated  watery  solution  .  5  c.c. 
Alum,  saturated  watery  solution  .  .  .  .5  c.c. 
Carbol-fuchsin  (vide  p.  104) 5  c.c. 

Mix  thoroughly.  A  precipitate  forms,  which  must  be  allowed  to  deposit, 
either  by  centrifugalising  or  simply  by  allowing  to  stand.  Remove  the  clear 
fluid  with  a  pipette  and  transfer  to  a  clean  bottle.  The  mordant  keeps  well 
for  one  or  two  weeks. 

B.  The  Stain. 

Alum,  saturated  watery  solution    .         .         .         .         .10  c.c. 
Gentian-violet,  saturated  alcoholic  solution  .         .         .       2  c.c. 

The  stain  should  not  be  more  than  two  or  three  days  old  when  used.  It  may 
be  substituted  in  the  mordant  in  place  of  the  carbol-fuchsin. 

The  film  having  been  prepared  as  above  described,  pour  over  it  as  much 
of  the  mordant  as  the  cover-glass  will  hold.  Heat  gently  over  a  flame  till 
steam  begins  to  rise,  allow  to  steam  for  about  a  minute,  and  then  wash  well  in 
a  stream  of  running  water  for  about  two  minutes.  Then  dry  carefully  over  the 
flame,  and  when  thoroughly  dry  pour  on  some  of  the  stain.  Heat  as  before, 
allowing  to  steam  for  about  a  minute,  wash  well  in  water,  dry,  and  mount  in  a 
drop  of  xylol -balsam. 

This  method  has  yielded  the  best  results  in  our  hands. 

2.  Loffler^s  Method. 
Two  solutions  must  be  made  up  as  follows :  — 

A.  The  Mordant* 

Tannic  acid,  20  per  cent  aqueous  solution  .  .  10  c.c. 
Ferrous  sulphate,  cold  saturated  aqueous  solution  .  5  c.c. 
Fuchsin,  saturated  alcoholic  solution  i  c.c. 

Mix  well,  set  aside  for  a  few  days  and  filter  always  before  using.  This  mor- 
dant improves  with  age. 

B.  The  Stain.  —  Either  carbol-fuchsin  or  aniline  gentian-violet  will  be 
found  to  be  eminently  satisfactory  if  filtered  before  using. 

Make  a  film  as  above  described,  and  holding  the  cover-glass  in  a  pair  of 
forceps,  pour  on  as  much  of  the  mordant  A  as  the  cover-glass  will  hold.  Heat 
it  carefully  above  a  flame  till  steam  begins  to  rise  and  then  move  the  prepara- 
tion gently  in  and  out  of  the  hot-air  column  over  the  flame  for  about  2  minutes. 
Wash  well  in  distilled  water  till  every  trace  of  mordant  appears  to  be  gone. 


THE    STAINING   OF   FLAGELLA.  IOQ 

If  necessary,  wash  with  absolute  alcohol  till  only  the  film  itself  appears  tinted 
violet  with  the  mordant.  Filter  a  few  drops  of  stain  B  on  to  the  cover,  again 
heat  till  steam  rises  and  leave  in  the  warm  stain  for  2  minutes.  Wash  well 
in  distilled  water,  dry,  and  mount  in  xylol-balsam. 

3.    Van  Ermengenfs  Method. 

The  films  are  prepared  as  above  described.  Three  solutions  are  here 
necessary :  — 

Solution  A.  (Bainfixateur)  — 

Osmic  acid,  2  per  cent  solution i  part. 

Tannin,  10-25  per  cent  solution          ....         2  parts. 

Place  the  films  in  this  for  one  hour  at  room  temperature,  or  heat  over  a 
flame  till  steam  rises  and  keep  in  the  hot  stain  for  five  minutes.  Wash  with 
distilled  water,  then  with  absolute  alcohol  for  three  to  four  minutes,  and  again 
in  distilled  water,  and  treat  with 

Solution  B.  (Bain  sensibilisateur)  — 

.5  per  cent  solution  of  nitrate  of  silver  in  distilled  water.     Allow  films 
to  be  in  this  a  few  seconds.     Then  without  washing  transfer  to 

Solution  C.  (Bain  reducteur  et  reinforqateur)  — 

Gallic  acid 5  grms. 

Tannin 3      ,, 

Fused  potassium  acetate 10      „ 

Distilled  water 350  c.c. 

Keep  in  this  for  a  few  seconds.  Then  treat  again  with  solution  B  till  the 
preparation  begins  to  turn  black.  Wash,  dry,  and  mount. 

It  is  better,  as  Mervyn  Gordon  recommends,  to  leave  the  specimen  in  B 
for  two  minutes  and  then  to  transfer  to  C  for  one  and  a  half  to  two  minutes, 
and  not  to  transfer  again  to  B.  It  will  also  be  found  an  advantage  to  use  a 
fresh  supply  of  C  for  each  preparation,  a  small  quantity  being  sufficient.  The 
beginner  will  find  the  typhoid  bacillus  or  the  bacillus  coli  communis  very  suita- 
ble organisms  to  stain  by  this  method. 

Although  the  results  obtained  by  this  method  are  sometimes  excellent, 
they  vary  considerably.  Frequently  both  the  organisms  and  flagella  appear 
of  abnormal  thickness.  This  is  due  to  the  fact  that  the  process  on  which  the 
method  depends  is  a  precipitation  rather  than  a  true  staining.  The  pictures 
on  the  whole  are  less  faithful  than  in  the  first  method. 

THE  TESTING  OF  AGGLUTINATIVE  AND  SEDIMENTING 
PROPERTIES  OF  SERUM. 

By  agglutination  is  meant  the  aggregation  into  clumps  of 
uniformly  disposed  bacteria  in  a  fluid,  by  sedimentation  the 
formation  of  a  deposit  composed  of  such  clumps  when  the 


110  MICROSCOPIC   METHODS. 

fluid  is  allowed  to  stand.  Sedimentation  is  thus  the  naked-eye 
evidence  of  agglutination.  The  blood  serum  may  acquire  this 
clumping  power  towards  a  particular  organism  under  certain 
conditions;  these  being  chiefly  met  with  when  the  individual  is 
suffering  from  the  disease  produced  by  the  organism,  or  has 
recovered  from  it,  or  when  a  certain  degree  of  immunity  has 
been  produced  artificially  by  injections  of  the  organism.  The 
nature  of  this  property  will  be  discussed  later.  Here  we  shall 
only  give  the  technique  by  which  the  presence  or  absence  of 
the  property  may  be  tested.  There  are  two  chief  methods,  a 
microscopic  and  a  naked  eye,  corresponding  to  the  effects  men- 
tioned above.  In  both,  the  essential  process  is  the  bringing  of 
the  diluted  serum  into  contact  with  the  bacteria  uniformly  dis- 
posed in  a  fluid.  In  the  former  this  is  done  on  a  glass  slide, 
and  the  result  is  watched  under  the  microscope;  the  occurrence 
of  the  phenomenon  is  shown  by  the  aggregation  of  the  bacteria 
into  clumps,  and  if  the  organism  is  motile  this  change  is  pre- 
ceded or  accompanied  by  more  or  less  complete  loss  of  motility. 
In  the  latter  method  the  mixture  is  placed  in  an  upright  thin 
glass  tube;  sedimentation  is  shown  by  the  formation  within  a 
given  time  (say  12  or  24  hours)  of  a  somewhat  flocculent  layer 
at  the  bottom,  the  fluid  above  being  clear.  Two  points  should 
be  attended  to;  (a)  controls  should  always  be  made  with  normal 
serum,  and  (£)  the  serum  to  be  tested  should  never  be  brought 
in  the  undiluted  condition  into  contact  with  the  bacteria.  The 
stages  of  procedure  are  the  following  :  — 

1.  Blood  is  conveniently  obtained  by  pricking  the  lobe  of  the  ear,  which 
should  previously  have  been  washed  with  a  mixture  of  alcohol  and  ether  and 
allowed  to  dry.     The  blood  is  drawn  up  into  the  bulbous  portion  of  a  capillary 
pipette,  such  as  in  Fig.  53,  a.      (These  pipettes  can  be  readily  made  by  draw- 
ing out  quill  glass  tubing  in  a  flame.     It  is  convenient  always  to  have  several 
ready  for  use.)     The  pipette  is  kept  in  the  upright  position,  one  end  being 
closed.     For  purposes  of  transit,  break  off  the  bulb  at  the  constriction  and 
seal  the  ends.     After  the  serum  has  separated  from  the  coagulum  the  bulb 
is  broken  through  near  its  upper  end,  and  the  serum  removed  by  means  of 
another  capillary  pipette.     The  serum  is  then  to  be  diluted. 

2.  The  serum  may  be  diluted  (a)  by  means  of  a  graduated  pipette  — 
either  a  leucocytometer  pipette  (Fig.  53,  b)  or  some  corresponding  form.     In 
this  way  successive  dilutions  of  i-io,  1-20,  i-ioo,  etc.,  can  be  rapidly  made. 
This  is  the  best  method.     (£)  By  means  of  a  capillary  pipette  with  a  mark  on 
the  tube  the  serum  is  drawn  up  to  the  mark  and  then  blown  out  into  a  glass 
capsule ;  equal  quantities  of  bouillon  are  successively  measured  in  the  same 


TESTING   AGGLUTINATIVE   POWER   OF    SERUM. 


Ill 


way  and  added  till  the  requisite  dilution  is  obtained.  (c}  By  means  of  a 
platinum  needle  with  a  loop  at  the  end  (Deldpine's  method).  A  loopful  of 
serum  is  placed  on  a  slide,  and  the  desired  number  of  similar  loopfuls  of 
bouillon  are  separately  placed  around  on  the  slide.  The  drops  are  then  mixed. 
A  very  convenient  and  rapid  method  of  combining  the  steps  i  and  2  is  to 
draw  a  drop  of  blood up  to  the  mark  i  or  .5  on  a  leucocytometer  pipette  and 
draw  the  bouillon  after  it  till  the  bulb  is  filled.  A  dilution  of  10  or  20  times 
is  thus  obtained.  Then  blow  the  mix- 
ture into  a  U-shaped  tube  (Fig.  53,  c) 
and  centrifugalise  or  simply  allow  the 
red  corpuscles  to  separate  by  stand- 
ing. (In  this  method  of  course  the 
dilution  is  really  greater  than  if  pure 
serum  were  used,  and  allowance  must 
therefore  be  made  in  comparing  re- 
sults.) The  presence  of  red  corpus- 
cles is  no  drawback  in  the  case  of 
the  microscopic  method,  but  when 
sedimentation  tubes  are  used  the  cor- 
puscles should  be  separated  first. 

3.  The    bacteria   to    be    tested 
should  be  taken  from  young  cultures, 
preferably  not  more  than  twenty-four 
hours  old,  incubated  at  37°  C.    They 
may  be  used  either  as   a   bouillon 
culture  or  as  an  emulsion  made  by 
adding  a  small  portion  of  an  agar 
culture   to   bouillon.      In   the  latter 
case  the  mass  of  bacteria  on  a  plati- 
num loop  shoujd  be  gently  broken 
down  at  the  margin  of  the  fluid  in  a 
watch-glass.    When  a  thick  turbidity 
is  thus  obtained,  any  remaining  frag- 
ments should  first  be  removed  and 
then  the  organisms  should  be  uni- 
formly mixed  with   the  rest  of  the 
fluid.     The  bacterial  emulsion  ought 
to  have  a  faint  but  distinct  turbidity. 
(When  the  exact  degree  of  sediment- 
ing  power  of  a  serum  is  to  be  tested  —  expressed  as  the  highest  dilution  in 
which  it  produces  complete  sedimentation  within  twenty-four  hours  —  a  stand- 
ard quantity  (by  weight)  of  bacteria  must  be  added  to  a  given  quantity  of 
bouillon.     This  is  not  necessary  for  clinical  diagnosis.) 

4.  To  test  microscopically,  mix  equal  quantities  (measured  by  a  marked 
capillary  pipette)  of  the  diluted  serum  and  the  bacterial  emulsion  on  a  glass 
slide,  cover  with  a  cover-glass  and  examine  under  the  microscope.    The  form  of 
glass  slide  used  for  hanging-drop  cultures  (Fig.  34)  will  be  found  very  suitable. 
The  ultimate  dilution  of  the  serum  will,  of  course,  be  double  the  original  dilution. 


FIG.  53.  —  Tubes  used  in  testing  agglutinating 
and  sedimenting  properties  of  serum. 


112  MICROSCOPIC   METHODS. 

To  observe  sedimentation  mix  equal  parts  of  diluted  serum  and  of  bacterial 
emulsion  and  place  in  a  thin  glass  tube  —  a  simple  tube  with  closed  end  or 
a  U-tube.  Keep  in  upright  position  for  twenty-four  hours.  One  of  W  right's 
sedimentation  tubes  is  shown  in  Fig.  53,  d.  Diluted  serum  is  drawn  up  to 
fill  the  space  »/»,  a  small  quantity  of  air  is  sucked  up  after  it  to  separate  it 
from  the  bacterial  emulsion,  which  is  then  drawn  up  in  the  same  quantity ; 
the  diluted  serum  will  then  occupy  the  position  kl.  The  fluids  are  then 
drawn  several  times  up  into  the  bulb  and  returned  to  the  capillary  tube  so 
as  to  mix,  and  finally  blown  carefully  down  close  to  the  lower  end,  which  is 
then  sealed  off.  The  sediment  collects  at  the  lower  extremity. 

GENERAL  BACTERIOLOGICAL  DIAGNOSIS. 

Under  this  heading  we  have  to  consider  the  general  routine 
which  is  to  be  observed  by  the  bacteriologist  when  any  material 
is  submitted  to  him  for  examination.  The  object  of  such 
examination  may  be  to  determine  whether  any  organisms  are 
present,  and  if  so,  what  organisms ;  or  the  bacteriologist  may 
simply  be  asked  whether  a  particular  organism  is  or  is  not  pres- 
ent. In  any  case  his  inquiry  must  consist  (i)  of  a  microscopic 
examination  of  the  material  submitted ;  (2)  of  an  attempt  to 
isolate  the  organisms  present ;  and  (3)  of  the  identification  of  the 
organisms  isolated.  We  must,  however,  before  considering  these 
points  look  at  a  matter  often  neglected  by  those  who  seek  a 
bacteriological  opinion,  viz.  :  the  proper  methods  of  obtaining  and 
transferring  to  the  bacteriologist  the  material  which  he  is  to  be 
asked  to  examine.  The  general  principles  here  are  (i)  that  every 
precaution  must  be  adopted  to  prevent  the  material  from  being 
contaminated  with  extraneous  organisms;  (2)  that  nothing  be 
done  which  may  kill  any  organisms  which  may  be  proper  to  the 
inquiry ;  and  (3)  that  the  bacteriologist  obtain  the  material  as 
soon  as  possible  after  it  has  been  removed  from  its  natural 
surroundings. 

The  sources  of  materials  to  be  examined,  even  in  patho- 
logical bacteriology  alone,  are  of  course  so  varied  that  we  can 
but  mention  a  few  examples.  It  is,  for  instance,  often  necessary 
to  examine  the  contents  of  an  abscess.  Here  the  skin  must  be 
carefully  purified  by  the  usual  surgical  methods  ;  the  knife  used 
for  the  incision  is  preferably  to  be  sterilised  by  boiling,  the  first 
part  of  the  pus  which  escapes  allowed  to  flow  away  (as  it  might 
be  spoiled  by  containing  some  of  the  antiseptics  used  in  the  purifi- 
cation) and  a  little  of  what  subsequently  escapes  allowed  to  flow 


REMARKS   ON   GENERAL   PROCEDURE. 


into  a  sterile  test-tube.  If  test-tubes  sterilised  in  a  laboratory 
are  not  at  hand,  an  ordinary  test-tube  may  be  a  quarter  filled 
with  water,  which  is  then  well  boiled  over  a  spirit  lamp.  The 
tube  is  then  emptied  and  plugged  with  a  plug  of  cotton  wool, 
the  outside  of  which  has  been  singed  in  a  flame.  Small  stop- 
pered bottles  may  be  sterilised  and  used  in  the  same  way.  A 
discharge  to  be  examined  may  be  so  small  in  quantity  as  to  make 
the  procedure  described  impracticable.  It  may  be  caught  on  a 
piece  of  sterile  plain  gauze,  or  of  plain  absorbent  wool,  which  is 
then  placed  in  a  sterile  vessel.  Wool  or  gauze  used  for  this  pur- 
pose, or  for  swabbing  out,  say  the  throat,  to  obtain  shreds  of 
suspicious  matter,  must  have  no  antiseptic  impregnated  in  it,  as 
the  latter  may  kill  the  bacteria  present  and  make  the  obtaining 
of  cultures  impossible. 

Fluids  from  the  body  cavities,  urine,  etc.,  may  be  secured 
with  sterile  pipettes.  To  make  one  of  these,  take  nine  inches 
of  ordinary  quill  glass  tubing,  draw  out  one  end  to  a  capillary 
diameter,  and  place  a  little  plug  of  cotton  wool  in 
the  other  end.  Insert  this  tube  through  the  cotton 
plug  of  an  ordinary  test-tube  and  sterilise  by  heat. 
To  use  it,  remove  test-tube  plug  with  the  quill 
tube  in  its  centre,  suck  up  some  of  the  fluid  into 
the  latter,  and  replace  in  its  former  position  in 
the  test-tube.  (Fig.  54.)  Another  method  very 
convenient  for  transport  is  to  make  two  constric- 
tions on  the  glass  tube  at  suitable  distances, 
according  to  the  amount  of  fluid  to  be  taken. 
The  fluid  is  then  drawn  up  into  the  part  between 
the  constrictions,  but  so  as  not  to  fill  it  com- 
pletely. The  tube  is  then  broken  through  at 
both  constrictions  and  the  thin  ends  are  sealed 

'*-~Xfr 

by  heating  in  a  flame.  FIG.  54.— Test- 

o    T  i  ,  ,      ,         ,  ,     ...  .      tube    and    pipette 

Solid  organs  to  be  examined  should,  if  possi-  arranged  for  ob- 
ble,  be  obtained  whole.     They  may  be  treated  in  taining  fluids  con- 

r  taining  bacteria. 

one  of  two  ways.  i.  The  surface  over  one  part 
about  an  inch  broad  is  seared  with  a  cautery  heated  to  dull  red 
heat.  All  superficial  organisms  are  thus  killed.  An  incision 
is  made  in  this  seared  zone  with  a  sterile  scalpel,  and  small 
quantities  of  the  juice  are  removed  by  a  platinum  loop  to  make 
cover-glass  preparations  and  plate  or  smear  cultures.  2.  An 


40; 


114  MICROSCOPIC   METHODS. 

alternative  method  is  as  follows :  The  surface  is  sterilised  by 
soaking  it  well  with  I  to  1000  corrosive  sublimate  for  half  an 
hour.  It  is  then  dried,  and  the  capsule  of  the  organ  is  cut 
through  with  a  sterile  knife,  the  incision  being  further  deepened 
by  tearing.  In  this  way  a  perfectly  uncontaminated  surface  is 
obtained.  Hints  are  often  obtained  from  the  clinical  history  of 
the  case  as  to  what  the  procedure  ought  to  be  in  examination. 
Thus,  as  a  matter  of  practice,  cultures  of  tubercle  and  often  of 
glanders  bacilli  can  be  easily  obtained  only  by  inoculation  experi- 
ments. Typhoid  bacilli  need  hardly  be  looked  for  in  the  faeces 
after  the  first  ten  days  of  the  disease,  and  so  on. 

Routine  Procedure  in  Bacteriological  Examination  of  Material. 
—  In  the  case  of  a  discharge  regarding  which  nothing  is  known 
the  following  procedure  should  be  adopted:  (i)  Several  cover- 
glass  preparations  should  be  made.  One  ought  to  be  stained 
with  saturated  watery  methylene-blue,  one  with  a  stain  contain- 
ing a  mordant  such  as  Ziehl-Neelsen  carbol-fuchsin,  one  by 
Gram's  method.  (2)  (a)  Gelatin  plates  should  be  made  and  kept 
at  room  temperature,  (b)  a  series  of  agar  plates  or  successive 
strokes  on  agar  tubes  (p.  57)  should  be  made  and  incubated  at 
37°  C.  Method  (b)  of  course  gives  results  more  quickly.  If 
microscopic  investigation  reveals  the  presence  of  bacteria,  it  is 
well  to  keep  the  material  in  a  cool  place  till  next  day  when,  if 
no  growth  has  appeared  in  the  incubated  agar,  some  other  culture 
medium  (e.g.  blood  serum  or  agar  smeared  with  blood)  may  be 
employed.  If  growth  has  taken  place,  say  in  the  agar  plates,  one 
with  about  200  or  fewer  colonies  should  be  made  the  chief  basis 
for  research.  In  such  a  plate  the  first  question  to  be  cleared  up 
is  :  Do  all  the  colonies  present  consist  of  the  same  bacterium  ? 
The  shape  of  the  colony,  its  size,  the  appearance  of  the  margin, 
the  graining  of  the  substance,  its  colour,  etc.,  are  all  to  be  noted. 
One  precaution  is  necessary,  viz.,  it  must  be  noted  whether  the 
colony  is  on  the  surface  of  the  medium  or  in  its  substance,  as 
colonies  of  the  same  bacterium  may  exhibit  differences  according 
to  their  position.  The  arrangement  of  the  bacteria  in  a  surface 
colony  may  be  still  more  minutely  studied  by  means  of  impression 
preparations.  A  cover-glass  is  carefully  cleaned  and  sterilised  by 
passing  quickly  several  times  through  a  Bunsen  flame.  It  is  then 
placed  on  the  surface  of  the  medium  and  gently  pressed  down 
on  the  colony.  The  edge  is  then  raised  by  a  sterile  needle,  it  is 


MORPHOLOGICAL  AND  CULTURAL  CHARACTERS.   115 

seized  with  forceps,  dried  high  over  the  flame,  and  treated  as  an 
ordinary  cover-glass  preparation.  In  this  way  very  characteristic 
appearances  may  sometimes  be  noted  and  preserved,  as  in  the 
case  of  the  anthrax  bacillus.  The  colonies  on  a  plate  having 
been  classified,  a  microscopic  examination  of  each  group  may  be 
made  by  means  of  cover-glass  preparations,  and  tubes  of  gelatin 
and  agar  are  inoculated  from  each  representative  colony.  Each 
of  the  colonies  used  must  be  marked  for  future  reference,  pref- 
erably by  drawing  a  circle  round  it  on  the  under  surface  of  the 
plate  or  Petri's  dish  with  one  of  Faber's  wax  pencils,  a  number 
or  letter  being  added  for  easy  reference. 

The  general  lines  along  which  observation  is  to  be  made1  in 
the  case  of  a  particular  bacterium  may  be  indicated  as  follows  :  — 

1.  Microscopic  Appearances.  —  For  ordinary  descriptive  pur- 
poses, young  cultures,  say  of  24  hours'  growth,  on  agar  should 
be  used,  though  appearances  in  older  cultures,  such  as  involu- 
tion forms,  etc.,  may  also  require  attention.     Note  (i)  the  form, 
(2)  the  size,  (3)  the  appearance  of  the  protoplasmic  contents, 
especially    as   regards   uniformity   or   irregularity   of   staining, 
(4)  the  method  of  grouping,  (5)  the  staining  reactions.     Has  it 
a  capsule  ?     Does  the  bacterium  stain  with  simple  watery  solu- 
tions?    Does  it  require  the  use  of  stains  containing  mordants? 
How  does  it  behave  towards  Gram's  method  ?     It  is  important 
to  investigate  the  first  four  points  both  when  the  organism  is  in 
the  fluids  or  tissues  of  the  body  and  when  growing  in  artificial 
media,  as  slight  variations  occur.     It  must  also  be  borne  in  mind 
that  slight  variations  are  observed,  according  to  the  kind  an'd 
consistence  of  the  medium  in  which  the  organism  is  growing. 
(6)  Is  it   motile,   and   has   it   flagella  ?     If   so,   how  are   they 
arranged?     (7)  Does  it   form    spores,   and   if   so,  under  what 
conditions  as  to  temperature,  etc.  ? 

2.  Growth  Characteristics.  —  Here  the  most  important  points 
on  which  information  is  to  be  asked  are,  What  are  the  charac- 
ters of  growth,  and  what  are  the  relations  of  growth  (i)  to  tem- 
perature, (2)  to  oxygen  ?     These  can  be  answered  from  some  of 
the  following  experiments  :  — 

1  The  student  is  asked  to  consult  the  recommendations  of  the  Bacteriological 
Committee  of  the  American  Public  Health  Association,  and  also  Chester's  "  A  Manual 
of  Determinative  Bacteriology,"  where  much  greater  detail  is  given  than  is  permissible 
\vithin  the  size  and  scope  of  this  work. 


Il6  MICROSCOPIC   METHODS. 

A.  Growth  on  gelatin,     (i)  Stab-culture.     Note  (a)  rate  of 
growth ;  (b)  form  of  growth,  (a)  on  surface,  (@)  in  substance ; 
(c)  presence  or  absence  of  liquefaction  ;  (d)  colour ;  (e)  presence 
or  absence  of  gas  formation  and  of  characteristic  smell ;  (/)  rela- 
tion  to   reaction   of   medium.     (2)  Streak-culture.     (3)  Shake- 
culture.      (4)  Plate-cultures.      Note    appearances    of    colonies 
(a)  superficial,  (b}  deep.      (5)  Growth  in  fluid  gelatin  at  37°  C. 

B.  Growth  on  agar  at  37°  C.     (i)  Stab.     (2)  Streak.     Also 
on  glycerin  agar,  blood  agar,  etc.     Appearances  of  colonies  in 
agar  plates. 

C.  Growth  in  bouillon,     (a)  Character  of  growth,  (b)  smell, 
(c)  reaction. 

D,.  Growth  on  special  media,  (i)  Solidified  blood  serum. 
(2)  Potatoes.  (3)  Lactose  and  other  sugar  media.  Does  fer- 
mentation occur,  and  is  gas  formed  ?  (4)  Milk.  Is  it  curdled 
or  turned  sour?  (5)  Litmus  media.  Note  changes  in  colour. 
(6)  Peptone  solution.  Is  indol  formed  ? 

E.   What  is  the  viability  of  organism  on  artificial  media  ? 

3.    Results  of  inoculation  experiments  on  animals. 

By  attention  to  such  points  as  these  a  considerable  know- 
ledge is  attained  regarding  the  bacterium,  which  will  lead  to  its 
identification.  In  the  case  of  many  well-known  organisms,  how- 
ever, a  few  of  the  above  points  taken  together  will  often  be  suffi- 
cient for  the  recognition  of  the  species,  and  experience  teaches 
what  are  the  essential  points  as  regards  any  individual  organism. 
In  the  course  of  the  systematic  description  of  the  pathogenic 
organisms,  it  will  be  found  that  all  the  above  points  will  be 
referred  to,  though  not  in  every  case. 

The  methods  by  which  the  morphological  and  biological  characteristics  of 
any  growth  may  be  observed  have  already  been  fully  described.  It  need  only 
be  pointed  out  here  that  in  giving  descriptions  of  bacteria  the  greatest  care 
must  be  taken  to  state  every  detail  of  investigation.  Thus  in  any  description 
of  microscopic  appearances  the  age  of  the  growth  from  which  the  preparation 
was  made,  the  medium  employed,  the  temperature  at  which  development  took 
place,  must  be  noted,  along  with  the  stain  which  was  used  ;  and  with  regard  to 
the  latter  it  is  always  preferable  to  employ  one  of  the  well-known  staining 
combinations,  such  as  Loffler's  methylene-blue.  Especial  care  is  necessary  in 
stating  the  size  of  a  bacterium.  The  apparent  size  often  shows  slight  variations 
dependent  on  the  stain  used  and  the  growth  conditions  of  the  culture.  Accurate 
measurements  of  bacteria  can  only  be  made  by  preparing  microphotographs  of 
a  definite  magnification  and  measuring  the  sizes  on  the  negatives.  From  these 


INOCULATION   OF   ANIMALS.  117 

the  actual  sizes  can  easily  be  calculated.  In  describing  bacterial  cultures  it 
must  be  borne  in  mind  that  the  appearances  often  vary  with  the  age.  It  is 
suggested  that  in  the  case  of  cultures  grown  at  from  36°  to  37°  C.  the  appear- 
ances between  24  and  48  hours  should  be  made  the  basis  of  description,  and 
in  the  case  of  cultures  grown  between  18°  and  22°  C.  the  appearances  between 
48  and  72  hours  should  be  employed.  The  culture  fluids  used  must  be  made 
up  and  neutralised  by  the  precise  methods  already  described.  The  investiga- 
tor must  give  every  detail  of  the  methods  he  has  employed  in  order  that  his 
observations  may  be  capable  of  repetition. 

INOCULATION  OF  ANIMALS 

The  animals  generally  chosen  for  inoculation  are  the  mouse, 
the  rat,  the  guinea-pig,  the  rabbit,  and  the  pigeon.  Great  cau- 
tion must  be  shown  in  drawing  conclusions  from  isolated  experi- 
ments on  rabbits,  as  these  animals  often  manifest  exceptional 
symptoms,  and  are  very  easily  killed.  Dogs  are,  as  a  rule, 
rather  insusceptible  to  microbic  disease,  and  the  larger  animals 
are  too  expensive  for  ordinary  laboratory  purposes.  In  the  case 
of  the  mouse  and  rat  the  variety  must  be  carefully  noted,  as 
there  are  differences  in  susceptibility  between  the  wild  and  tame 
varieties,  and  between  the  white  and  brown  varieties  of  the  latter. 
In  the  case  of  the  wild  varieties,  these  must  be  kept  in  the  labo- 
ratory for  a  week  or  two  before  use,  as  in  captivity  they  are 
apt  to  die  from  very  slight  causes,  and,  further,  each  individual 
should  be  kept  in  a  separate  cage,  as  they  show  great  tenden- 
cies to  cannibalism.  Of  all  the  ordinary  animals  the  most  sus- 
ceptible to  microbic  disease  is  the  guinea-pig.  Practically  all 
inoculations  are  performed  by  means  of  the  hypodermic  syringe. 
The  best  variety  is  made  on  the  ordinary  model  with  metal  mount- 
ings, asbestos  washers,  and  preferably  furnished  with  platinum 
indium  needles.  Before  use  the  syringe  and  the  needle  are 
sterilised  by  boiling  for  five  minutes.  The  materials  used  for 
inoculation  are  cultures,  animal  exudations,  or  the  juice  of  organs. 
If  the  bacteria  already  exist  in  a  fluid  there  is  no  difficulty.  The 
syringe  is  most  conveniently  filled  out  of  a  shallow  conical  test- 
glass  which  ought  previously  to  have  been  covered  with  a  cover 
of  filter  paper  and  sterilised.  If  an  inoculation  is  to  be  made 
from  organisms  growing  on  the  surface  of  a  solid  medium, 
either  a  little  ought  to  be  scraped  off  and  shaken  up  in  sterile 
distilled  water  or  75  per  cent  salt  solution  to  make  an  emulsion, 
or  a  little  sterile  fluid  is  poured  on  the  growth  and  the  latter 


Il8  MICROSCOPIC   METHODS. 

scraped  off  into  it.  This  fluid  is  then  filtered  into  the  test-glass 
through  a  plug  of  sterile  glass  wool.  This  is  easily  effected  by 
taking  a  piece  of  -|  in.  glass  tubing  3  in.  long,  drawing  one  end 
out  to  a  fairly  narrow  point,  plugging  the  tube  with  glass  wool 
above  the  point  where  the  narrowing  commences,  and  sterilis- 
ing by  heat.  By  filtering  an  emulsion  through  such  a  pipette, 
flocculi  which  might  block  the  needle  are  removed.  If  a  solid 
organ  or  an  old  culture  is  used  for  inoculation  it  ought  to  be 
rubbed  up  in  a  sterile  porcelain  or  metal  crucible  with  a  little 
sterile  distilled  water,  by  means  of  a  sterile  glass  rod,  and  the 
emulsion  filtered  as  in  the  last  case. 

The  methods  of  inoculation  generally  used  are:  (i)  by 
scarification  of  the  skin;  (2)  by  subcutaneous  injection;  (3) 
by  intraperitoneal  injection;  (4)  by  intravenous  injection;  (5) 
by  injections  into  special  regions,  such  as  the  anterior  chamber 
of  the  eye,  the  substance  of  the  lung,  etc.  Of  these  (2),  (3), 
and  (4)  are  most  frequently  used.  When  an  anaesthetic  is 
to  be  administered,  this  is  conveniently  done  by  placing  the 
animal,  along  with  a  piece  of  cotton  wool  or  sponge  soaked 
in  chloroform,  under  a  bell-jar  or  inverted  glass  beaker  of  suita- 
ble size. 

i.  Scarification.  —  A  few  parallel  scratches  are  made  in  the 
skin  of  the  abdomen  previously  cleansed,  just  sufficiently  deep 
to  draw  blood,  and  the  infective  material  is  rubbed  in  with  a 
platinum  eyelet.  The  disadvantage  of  this  method  is  that  the 

inoculation  is  easily  con- 
taminated. The  method 
is  only  occasionally  used. 
2.  Subcutaneous  Injec- 
tion.— A  hypodermic  syr- 
inge is  charged  with  the 
fluid  to  be  inoculated. 

FIG.  55.  — Apparatus  for  holding  a  mouse  prepara-    The    hair     is    CUt    off    the 
tory  to  subcutaneous  inoculation. 

part  to  be  inoculated,  and 

the'  skin  purified  with  i  to  1000  corrosive  sublimate.  The  skin 
is  then  pinched  up,  and,  the  needle  being  inserted,  the  requisite 
dose  is  administered.  The  wound  is  then  sealed  with  a  little 
collodion. 

3.  Intraperitoneal  Injection.  —  This  may  be  performed  by 
means  of  a  special  form  of  needle.  The  needle  is  curved,  and 


INTRAPERITONEAL   INOCULATION.  119 

has  its  opening,  not  at  the  point,  but  in  the  side  in  the  middle  of 
the  arch  (Fig.  56).  The  hair  over  the  lower  part  of  the  abdomen 
is  cut,  and  the  skin  purified  with  an  antiseptic.  The  whole  thick- 
ness of  the  abdominal  walls  is  then  pinched  up  by  an  assistant, 
between  the  forefingers  and  thumbs  of  the  two 
hands.  The  needle  is  then  plunged  through  the 
fold  thus  formed.  The  result  is  that  the  hole  in 
the  side  of  the  needle  is  within  the  abdominal 
cavity,  and  the  inoculation  can  thus  be  made. 
Intraperitoneal  inoculation  can  also  be  practised 
with  an  ordinary  needle.  The  mode  of  proced- 
ure is  similar,  but  after  the  needle  is  plunged 
through  the  abdominal  fold,  it  is  partially  with- 
drawn till  the  point  is  felt  to  be  free  in  the  peri-  FIG.  56.  —  Hollow 
toneal  cavity,  when  the  injection  is  made.  There  aperture^at^fon?- 
is  little  risk  of  injuring  the  intestines  by  either  trapedtoneai  inocu- 

,  i       i  lations. 

method. 

4.  Intravenous  Injection.  —  The  vein  most  usually  chosen  is 
one   of   the   auricular   veins ;   preferably   the   posterior   lateral 
branch.     The  part  has  the  hair  removed,  the  skin  is  purified, 
and  the  vein  made  prominent  by  pressing  on  it  between  the 
point  of  inoculation  and  the  heart.     The  needle  is  then  plunged 
into  the  vein  and  the  fluid  injected.      That  it  has  perforated  the 
vessel  will  be  shown  by  the  escape  of  a  little  blood ;  and  that 
the  injection  has  taken  place  into  the  lumen  of  the  vessel  will 
be  known  by  the  absence  of  the  small  swelling  which  occurs  in 
subcutaneous  injections.      If  preferred,  the  vein  may  be  first 
laid  bare  by  snipping  the  skin  over  it.      The  needle  is  then 
introduced. 

5.  Inoculation  into  the  Anterior  Chamber  of  the  Eye.  —  Local 
anaesthesia  is  established  by  applying  a  few  drops  of  2  per  cent 
solution    of    hydrochlorate  of    cocaine.      The  eye  is  fixed  by 
pinching  up  the  orbital  conjunctiva  with  a  pair  of  fine  forceps, 
and  the  edge  of  the  cornea  being  perforated  by  the  hypodermic 
needle,  the  injection  is  easily  accomplished. 

Sometimes  inoculations  are  made  by  planting  small  pieces  of 
pathological  tissues  in  the  subcutaneous  tissue.  This  is  especially 
done  in  the  case  of  glanders  and  tubercle.  The  skin  over  the 
back  is  purified,  and  the  hair  cut.  A  small  incision  is  made  with 
a  sterile  knife,  and  the  skin  being  separated  from  the  subjacent 


120  MICROSCOPIC   METHODS. 

tissues  by  means  of  the  ends  of  a  blunt  pair  of  forceps,  a  little 
pocket  is  formed  into  which  a  piece  of  the  suspected  tissue  is 
inserted.  The  wound  is  then  closed  with  a  suture,  and  collodion 
is  applied.  In  the  case  of  guinea-pigs,  the  abdominal  wall  is  to 
be  preferred  as  the  site  of  inoculation,  as  the  skin  over  the  back 
is  extremely  thick. 

Injections  are  sometimes  made  into  other  parts  of  the  body, 
e.g.  the  pleurae  and  the  cranium.  It  is  unnecessary  to  describe 
these,  as  the  application  of  the  general  principles  employed 
above,  together  with  those  of  modern  aseptic  surgery,  will 
sufficiently  guide  the  investigator  as  to  the  technique  which  is 
requisite. 

After  inoculation,  the  animals  ought  to  be  kept  in  comfortable 
cages,  which  must  be  capable  of  easy  and  thorough  disinfection 
subsequently.  For  this  purpose  galvanised  iron  wire  cages  are 
the  be^t.  They  can  easily  be  sterilised  by  boiling  them  in  the 
large  fisi> -kettle  which  it  is  useful  to  have  in  a  bacteriological 
laboratory  for  such  a  purpose.  It  is  preferable  to  have  the 
cages  opening  from  above.  Otherwise  material  which  may  be 
infective  may  be  scratched  out  of  the  cage  by  the  animal.  The 
general  condition  of  the  animal  is  to  be  observed,  how  far  it 
differs  from  the  normal,  whether  there  is  increased  rapidity  of 
breathing,  etc.  The  temperature  is  usually  to  be  taken.  This 
is  generally  done  per  rectum.  The  thermometer  (the  ordinary 
5  min.  clinical  variety)  is  smeared  with  vaseline,  and  the  bulb 
inserted  just  within  the  sphincter,  where  it  is  allowed  to  remain 
for  a  minute ;  it  is  then  pushed  well  into  the  rectum,  again 
remaining  for  five  minutes.  If  this  precaution  be  not  adopted, 
a  reflex  contraction  of  the  vessels  may  take  place,  which  is  likely 
to  vitiate  the  result  by  giving  too  low  a  reading. 

Autopsies  on  Animals  dead  or  killed  after  Inoculation.  — 
These  should  be  made  as  soon  as  possible  after  death.  It  is 
necessary  to  have  some  shallow  troughs,  constructed  either  of 
metal  or  of  wood  covered  with  metal,  conveniently  with  sheet 
lead,  and  having  a  perforation  at  each  corner  to  admit  a  tape  or 
strong  cord.  The  animal  is  tightly  stretched  out  in  the  trough 
and  tied  in  position.  The  size  of  the  trough  will,  therefore,  have 
to  vary  with  the  size  of  the  outstretched  body  of  the  animal  to 
be  examined.  In  certain  cases  it  is  well  to  soak  the  surface  of 
the  animal  in  carbolic  acid  solution  (i  to  20),  or  in  corrosive 


AUTOPSIES    ON    ANIMALS.  121 

sublimate  (i  to  1000),  before  it  is  tied  out.  This  not  only  to  a 
certain  extent  disinfects  the  skin,  but,  what  is  more  important, 
prevents  hairs  which  might  be  affected  with  pathogenic  products 
from  getting  into  the  air  of  the  laboratory.  The  instruments 
necessary  are  scalpels  (preferably  with  metal  handles),  dissecting 
forceps,  and  scissors.  They  are  to  be  sterilised  by  boiling  for 
five  minutes.  This  is  conveniently  done  in  one  of  the  small 
portable  sterilisers  used  by  surgeons.  Two  sets  at  least  ought 
to  be  used  in  an  autopsy,  and  they  may  be  placed,  after  boiling, 
on  a  sterile  glass  plate  covered  by  a  bell-jar.  It  is  also  necessary 
to  have  a  medium-sized  hatchet-shaped  cautery,  or  other  similar 
piece  of  metal.  It  is  well  to  have  prepared  a  few  freshly  drawn- 
out  capillary  tubes  stored  in  a  sterile  cylindrical  glass  vessel,  and 
also  some  larger  sterile  glass  pipettes.  The  hair  of  the  abdomen 
of  the  animal  is  removed.  If  some  of  the  peritoneal  fluid  is 
wanted,  a  band  should  be  cauterised  down  the  linea  alba  Ifrom 
the  sternum  to  the  pubes,  and  another  at  right  angles' 'to  the 
upper  end  of  this ;  an  incision  should  be  made  in  the  middle  of 
these  bands,  and  the  abdominal  walls  thrown  to  each  side.  One 
or  more  capillary  tubes  should  then  be  filled  with  the  fluid  col- 
lected in  the  flanks,  the  fluid  being  allowed  to  run  up  the  tube 
and  the  point  sealed  off ;  or  a  larger  quantity,  if  desired,  is  taken 
in  a  sterile  pipette.  If  peritoneal  fluid  be  not  wanted,  then  an 
incision  may  be  made  from  the  episternum  to  the  pubes,  and  the 
thorax  and  abdomen  opened  in  the  usual  way.  The  organs 
ought  to  be  removed  with  another  set  of  instruments,  and  it  is 
convenient  to  place  them  pending  examination  in  sterilised  deep 
Petri's  dishes.  It  is  generally  advisable  to  make  cultures  and 
film  preparations  from  the  heart's  blood.  To  do  this,  open  the 
pericardium,  sear  the  front  of  the  right  ventricle  with  a  cautery, 
make  an  incision  in  the  middle  of  the  part  seared,  and  remove 
some  of  the  blood  with  a  capillary  tube  for  future  examination, 
or,  introducing  a  platinum  eyelet,  inoculate  tubes  and  make 
cover-glass  preparations1  at  once.  To  examine  any  organ,  sear 
the  surface  with  a  cautery,  cut  into  it,  and  inoculate  tubes  and 
make  film  preparations  with  a  platinum  loop.  For  removing 
small  parts  of  organs  for  making  inoculations  on  tubes,  a  small 
platinum  spud  is  very  useful,  as  the  ordinary  wires  are  apt  to 
become  bent.  Place  pieces  of  the  organs  in  some  preservative 
fluid  for  microscopic  examination.  The  organs  ought  not  to  be 


122  MICROSCOPIC   METHODS. 

touched  with  the  fingers.  When  the  post  mortem  is  concluded 
the  body  should  have  corrosive  sublimate  or  carbolic  acid  solution 
poured  over  it,  and  be  forthwith  burned.  The  dissecting  trough 
and  all  the  instruments  ought  to  be  boiled  for  half  an  hour.  The 
amount  of  precaution  to  be  taken  will,  of  course,  depend  on  the 
character  of  the  bacterium  under  investigation,  but  as  a  general 
rule  every  care  should  be  used. 


CHAPTER    IV. 

BACTERIA   IN   AIR,    SOIL,   AND   WATER. 
ANTISEPTICS. 

IT  is  impossible  here  to  do  more  than  indicate  the  chief 
methods  which  are  employed  by  bacteriologists  in  the  investiga- 
tion of  the  bacteria  present  in  air,  soil,  and  water,  and  to  add  an 
outline  of  the  chief  results  obtained.  In  dealing  with  the  latter 
the  subject  has  been  approached  mainly  from  the  standpoint  of 
the  bearings  which  the  results  have  towards  human  pathology. 
In  dealing  with  antiseptics,  so  far  as  possible  the  effects  of  the 
various  agents  on  the  chief  pathogenic  bacteria  have  been  given, 
though  in  many  cases  our  information  is  very  imperfect. 

Am. 

Very  little  information  of  value  can  be  obtained  from  the 
examination  of  the  air,  but  the  following  are  the  chief  methods 
used,  along  with  the  results  obtained.  More  can  be  learned 
from  the  examination  of  atmospheres  experimentally  contami- 
nated than  by  the  investigation  of  the  air  as  it  exists  under 
natural  conditions. 

Methods  of  Examination.  —  The  methods  employed  vary  with  the  objects 
in  view.  If  it  be  sought  to  compare  the  relative  richness  of  different  atmos- 
pheres in  organisms,  and  if  the  atmospheres  in  question  be  fairly  quiescent, 
then  it  is  sufficient  to  expose  gelatin  plates  for  definite  times  in  the  rooms  to 
be  examined.  Bacteria,  or  the  particles  of  dust  carrying  them,  fall  on  the 
plates,  and  from  the  number  of  colonies  which  develop  a  rough  idea  of  the 
richness  of  the  air  in  bacteria  can  be  obtained.  Petri  states  that  in  five  min- 
utes the  number  of  bacteria  present  in  10  litres  of  air  are  deposited  on  100 
square  centimetres  of  a  gelatin  plate. 

More  complete  results  are  available  when"  some  method  is  employed  by 
which  the  bacteria  in  a  given  quantity  of  air  are  examined.  The  oldest 
method  employed,  and  one  which  is  still  used,  is  that  of  Hesse.  The 
apparatus  is  shown  in  Fig.  57.  It  consists  of  a  cylindrical  tube  a  about 
20  inches  long  and  2  inches  in  diameter.  At  one  end  this  is  closed  by  a 
rubber  cork  having  a  piece  of  quill  tubing  f  passing  through  it  and  projecting 
some  distance  into  the  interior.  For  use  the  tube  is  sterilised  in  a  tall  Koch, 

123 


124 


BACTERIA   IN   AIR,  SOIL,  AND  WATER. 


and  then  a  quantity  of  peptone  gelatin,  sufficient  to  cover  the  whole  interior  to 
the  thickness  of  an  ordinary  gelatin  plate,  is  poured  in.  This  gelatin  is  kept 
from  escaping  by  the  projection  of  the  quill  tubing  into  the  lumen  of  the  large 
tube.  A  plug  of  cotton  wool  is  now  placed  in  the  outer  end  of  the  quill  tubing. 

Over  the  other  end  of  the  large  tube 
is  tied  a  sheet  of  rubber  having  a 
hole  about  a  quarter  of  an  inch  in 
diameter  in  its  centre,  and  over  this 
again  is  tied  a  piece  of  similar  but 
unperforated  sheet  rubber.  The  tube 
is  then  sterilised  in  the  tall  Koch. 
On  removal  from  this  it  is  rolled, 
after  the  manner  of  an  Esmarch's 
tube  (q.v.)  till  the  gelatin  is  set  as 
a  layer  over  its  interior,  and  it  is 
then  placed  horizontally  on  the 
tripod  as  shown.  The  other  part 
of  the  apparatus  is  an  aspirator  by 
means  of  which  a  known  quantity 
of  air  can  be  brought  in  contact 
with  the  gelatin.  It  consists  of  two 
conical  glass  flasks  connected  by 
means  of  a  tube  which  passes  through 
the  cork  of  each  down  to  the  bottom 
of  the  flask.  When  this  tube  is 
filled  with  water,  it,  of  course,  can  act 
as  a  syphon  tube  between  volumes 
of  water  in  the  flasks.  Such  a  syphon 
system  being  established,  the  levels  of  the  water  are  marked  on  the  flasks,  and 
to  one  a  litre  of  water  is  added,  and  by  depressing  flask  b  the  whole  litre  can 
be  got  into  it  and  the  connecting  tube  c  is  then  clamped.  The  two  flasks  are 
then  connected  by  a  rubber  tube  with  the  tube/,  the  clamp  on  c  is  opened,  and 
the  passing  of  a  litre  of  water  into  d  will  draw  a  litre  of  air  through  the  gelatin 
tube,  when  the  outer  rubber  sheet  is  removed  from  the  end  and  the  clamp  h 
opened.  By  disconnecting  at  g  and  reversing  the  syphon  flasks,  another  litre 
can  be  sucked  through,  and  so  any  desired  quantity  of  air  can  be  brought  in 
contact  with  the  gelatin.  The  speed  ought  not  to  be  more  than  i  litre  in 
two  minutes,  and  in  such  a  case  practically  all  the  organisms  will  be  found  to 
have  fallen  out  of  the  air  on  to  the  gelatin  in  the  course  of  their  transit.  This 
fact  can  be  tested  by  interposing  between  the  tube  a  and  the  aspirator  a  second 
tube  prepared  in  the  same  way,  which  ought,  of  course,  to  show  no  growth. 
When  forty-eight  hours  at  20°  C.  or  four  days  at  lower  temperature  have 
elapsed,  the  colonies  which  develop  in  a  may  be  counted.  The  disadvantage 
of  the  method  is  that  if  particles  of  dust  carrying  more  than  one  bacterium 
alight  on  the  gelatin,  these  bacteria  develop  in  one  colony,  and  thus  the  enu- 
meration results  may  be  too  low;  difficulties  may  also  arise  from  liquefying 
colonies  developing  in  the  upper  parts  of  the  tube  and  running  over  the 
gelatin. 


FIG.  57.  —  Hesse's  tube,  mounted  for  use. 


BACTERIOLOGICAL   EXAMINATION    OF   AIR. 


125 


.d 


FIG.  58.  —  Petri's 
sand  filters. 


Petri's  Sand-filter  Method. — A  glass  tube  open  at  both  ends,  and  about 
3^  inches  long  and  half  an  inch  wide,  is  taken,  and  in  its  centre  is  placed  a 
transverse  diaphragm  of  very  fine  iron  gauze  (Fig.  58,  e)  ;  on 
each  side  of  this  is  placed  some  fine  quartz  sand  which  has 
been  well  washed,  dried,  and  burned  to  remove  all  impurities, 
and  this  is  kept  in  position  by  cotton  plugs.  The  whole 
is  sterilised  by  dry  heat.  One  plug  is  removed  and  a  sterile 
rubber  cork  c  inserted,  through  which  a  tube  d  passes  to 
an  exhausting  apparatus.  The  tube  is  then  clamped  in  an 
upright  position  in  the  atmosphere  to  be  examined,  with 
the  remaining  plug  f  uppermost.  The  latter  is  removed 
and  the  air  sucked  through.  Difficulty  may  be  experienced 
from  the  resistance  of  the  sand  if  quick  filtration  be  at- 
tempted. The  best  means  to  adopt  is  to  use  an  air-pump 

—  the  amount  of  air  drawn  per 
stroke  of  which  is  accurately 
known — and  have  a  manometer 
(as  in  Fig.  38)  interposed  be- 
tween the  tube  and  the  pump. 
Between  each  two  strokes  of 
the  air-pump  the  mercury  is 
allowed  to  return  to  zero.  After 
the  required  amount  of  air  has  passed,  the  sand 
a  is  removed,  and  is  distributed  among  a  number 
of  sterile  gelatin  tubes  which  are  well  shaken ; 
plate-cultures  are  then  made,  and  when  growth 
has  occurred  the  colonies  are  enumerated ;  the 
sand  b  is  similarly  treated  and  acts  as  a  control. 
The  Sedgwick-Tucker  Method.  —  A  third  and 
better  method  is  that  of  Sedgwick  and  Tucker, 
whose  apparatus  combines  the  qualities  of  both 
filter  and  culture  tubes,  whilst  the  employment  of 
finely  granulated  sugar  as  the  filtering  medium 
removes  the  very  obvious  objections  to  the  use 
of  sand.  The  apparatus  consists  of  a  glass  tube 
some  30  or  35  cm.  long  and  4  cm.  wide,  drawn 
out  at  one  end  into  a  neck  to  retain  a  cotton 
plug,  whilst  into  the  other  end  is  fused  a  smaller 
piece  of  glass  tubing  about  15  cm.  long  and  .5  cm. 
wide.  The  surface  of  the  larger  portion  is  ruled 
by  a  diamond  into  square  centimetres  to  facili- 
tate the  counting  of  colonies  (see  Fig.  59).  To 
retain  the  filtering  medium  in  position,  a  small 
piece  of  tightly  rolled  fine-meshed  brass-wire 
FIG.  59.— The  Sedgwick-Tucker  cloth  is  inserted  into  this  narrow  tubing  about 
aerobioscope.  ^  cm  from  jts  iower  end.  After  cleansing  and 

drying,  the  bore  of  the  smaller  part  of  the  apparatus  is  filled  with   No.  50 
granulated  sugar,  which  is  lightly  packed  by  gently  tapping,  both  ends  are 


By  permission,  from  Abbott's 
"  Bacteriology." 


126  BACTERIA    IN   AIR,  SOIL,  AND   WATER. 

plugged  with  cotton,  and  the  filter  is  then  sterilised  for  two  or  three  hours  at 
I2O°C.  —  a  higher  temperature  is  liable  to  char  the  sugar.  When  used,  the 
filter  is  to  be  affixed  by  its  lower  end  to  the  aspirating  pump  and  kept  in  an 
upright  position,  the  upper  plug  of  cotton  is  now  removed  and  aspiration 
carried  out.  When  this  is  ended,  the  cotton  plug,  having  meantime  been 
placed  in  a  sterile  receptacle,  is  re-inserted,  the  apparatus  disconnected,  the 
sugar  by  gentle  rapping  transferred  to  the  upper  portion,  15  c.c.  of  sterile 
liquefied  gelatin  poured  into  the  filter,  the  filter  plugged,  and  the  sugar 
dissolved.  The  filter  is  now  to  be  treated  as  an  Esmarch  roll-tube,  by 
being  rolled  on  ice  until  the  gelatin  sets,  when  the  apparatus  is  set  aside 
at  room  temperature  for  incubation.  This  method  gives  very  accurate  re- 
sults. 

When  it  is  necessary  to  examine  air  for  particular  organisms,  special 
methods  must  often  be  adopted.  Thus  in  the  case  of  the  suspected  presence 
of  tubercle  bacilli  a  given  quantity  of  air  is  drawn  through  a  small  quantity  of 
water  and  then  injected  into  a  guinea-pig. 

It  must  be  admitted  that  comparatively  little  information 
bearing  on  the  harmlessness  or  harmfulness  of  the  air  is  obtain- 
able by  the  mere  enumeration  of  the  living  organisms  present, 
for  under  certain  conditions  the  number  may  be  increased  by  the 
presence  of  many  individuals  of  a  purely  non-pathogenic  charac- 
ter. The  organisms  found  in  the  air  belong  to  two  groups  —  firstly, 
a  great  variety  of  bacteria ;  secondly,  yeasts  and  the  spores  of 
moulds  and  of  the  lower  fungi.  With  regard  to  the  spores,  the 
organisms  from  which  they  are  derived  often  consist  of  felted 
masses  of  threads,  from  which  are  thrust  into  the  air  special 
filaments,  and  in  connection  with  these  the  spores  are  formed. 
By  currents  of  air  these  laU  .t  can  easily  be  detached,  and  may  float 
about  in  a  free  condition.  With  the  bacteria,  on  the  other  hand, 
the  case  is  different.  Usually  these  are  growing  together  in 
little  masses  on  organic  materials,  or  in  fluids,  and  it  is  only 
by  the  detachment  of  minute  particles  of  the  substratum  that 
the  organisms  become  free.  The*  entrance  of  bacteria  into  the 
air,  therefore,  is  associated  with  conditions  which  favour  the 
presence  of  dust,  minute  droplets  of  fluid,  etc.  The  presence 
of  dust  in  particular  would  specially  favour  a  large  number  of 
bacteria  being  observed,  and  this  is  the  case  with  the  air  in  many 
industrial  conditions,  where  the  bacteria,  though  numerous,  may 
be  quite  innocuous.  Great  numbers  of  bacteria  thus  may  not 
indicate  any  condition  likely  to  injure  health,  and  this  maybe 
true  also  even  when  the  bacteria  come  from  the  crowding  together 
of  a  number  of  healthy  human  beings.  On  the  other  hand, 


DISSEMINATION   OF   BACTERIA   IN   AIR.  127 

there  is  no  doubt  that  disease  germs  can  be  disseminated  by 
means  of  the  air.  The  possibility  of  this  has  been  shown  ex- 
perimentally by  infecting  the  mouth  with  the  B.  prodigiosus, 
which  is  easily  recognised  by  its  brilliantly  coloured  colonies, 
and  then  studying  its  subsequent  distribution.  Most  important 
here  is  the  infection  of  the  air  from  sick  persons.  The 
actions  of  coughing,  sneezing,  speaking,  and  even  of  deep 
breathing,  distribute,  often  to  a  considerable  distance,  minute 
droplets  of  secretions  from  the  mouth,  throat,  and  nose,  and  these 
may  float  in  the  air  for  a  considerable  time.  Even  five  hours 
after  an  atmosphere  has  been  thus  infected  evidence  may  be 
found  of  bacteria  still  floating  free.  Before  this  time,  however, 
most  of  the  bacteria  have  settled  upon  various  objects,  where 
they  rapidly  dry,  and  are  no  longer  displaceable  by  ordinary  air 
currents.  The  diseases  of  known  etiology  where  infection  can 
thus  take  place  are  diphtheria,  influenza,  pneumonia,  and  phthisis; 
and  here  also  probably  whooping-cough,  typhus  fever,  and  measles 
are  to  be  added,  though  the  morbific  agents  are  unknown.  In 
the  case  of  phthisis,  the  alighting  of  tubercle  bacilli  has  been 
demonstrated  on  cover-glasses  held  before  the  mouths  of  patients 
while  talking,  and  animals  made  to  breathe  directly  in  front  of 
the  mouths  of  such  patients  have  become  infected  with  tuber- 
culosis. Apart  from  direct  infection  from  individuals,  however, 
pathogenic  bacteria  may  be  spread  in  some  cases  from  the  splash- 
ing of  infected  water,  as  from  a  sewage  outfall.  This  possibility 
has  to  be  recognised  especially  in  .  j  cases  of  typhoid  and 
cholera.  Besides  infection  through  fluid  particles,  infection  can 
be  caused  in  the  air  by  dust  coming,  say,  from  infected  skin  or 
clothes,  etc.  Fliigge,  in  dealing  with  this  subject  in  an  experi- 
mental inquiry,  distinguishes  between  large  particles  of  dust 
which  require  an  air  current  moving  at  the  rate  of  i  cm.  per 
second  to  keep  them  suspended,  and  the  finer  dust  which  can 
be  kept  in  suspension  by  currents  moving  at  from  i  to  4  mm. 
per  second.  In  the  former  case,  when  once  the  particles  alight 
they  cannot  be  displaced  by  currents  of  air  except  when  these 
are  moving  at,  at  least,  5  m.  per  second,  but  the  brushing, 
shaking,  or  beating  of  objects  may,  of  course,  distribute  them. 
In  the  case  of  the  finer  dust  the  particles  will  remain  for  long 
suspended,  and  when  they  have  settled  can  be  more  easily  dis- 
placed, as  by  the  waving  of  an  arm,  breathing,  etc.  With  regard 


128  BACTERIA   IN    AIR,  SOIL,  AND    WATER. 

to  infection  by  dust,  a  most  important  factor,  however,  is  whether 
or  not  the  infecting  agent  can  preserve  its  vitality  in  a  dry  con- 
dition. In  the  case  of  a  sporing  organism  such  as  anthrax, 
vitality  is  preserved  for  long  periods  of  time,  and  great  resist- 
ance to  drying  is  also  possessed  by  the  tubercle  and  diphtheria 
bacilli ;  but  apart  from  such  cases  there  is  little  doubt  that  infec- 
tion is  usually  necessarily  associated  with  the  transport  of  most 
particles,  and  is  thus  confined  to  a  limited  area  around  a  sick 
person.  Among  diseases  which  may  occasionally  be  thus  spread 
cholera  and  typhoid  have  been  classed.  Considerable  contro- 
versy has  arisen  with  regard  to  certain  outbreaks  of  the  latter 
disease,  which  have  apparently  been  spread  by  dusty  winds, 
although  we  have  the  fact  that  the  typhoid  bacillus  does  not 
survive  being  dried  even  for  a  short  time.  It  appears,  however, 
that  in  such  epidemics  the  transport  of  infection  by  means  of 
insects  carried  by  the  wind  has  not  been  entirely  excluded ;  in 
fact,  the  common  house-fly  comes  strongly  under  suspicion  of 
being  the  carrier  of  infection  during  epidemics  of  typhoid  fever 
and  cholera,  where  fecal  discharges  may  have  been  carelessly 
disposed  of. 

SOIL. 

The  investigation  of  the  bacteria  which  may  be  found  in  the 
soil  is  undertaken  from  various  points  of  view.  Information 
may  be  desired  as  to  the  change  its  composition  undergoes  by 
a  bacterial  action,  the  result  of  which  may  be  an  increase  in 
fertility  and  thus  in  economic  value.  Under  this  head  may  be 
grouped  inquiries  relating  to  the  bacteria  which  convert  am- 
monia and  its  salts  into  nitrates  and  nitrites,  and  to  the  organ- 
isms concerned  in  the  fixation  of  the  free  nitrogen  of  the  air. 
The  discussion  of  the  questions  involved  in  such  inquiries  is 
outside  the  scope  of  the  present  chapter,  which  is  more  con- 
cerned with  the  relation  of  the  bacteriology  of  the  soil  to  ques- 
tions of  public  health.  So  far  as  this  narrower  view  is  con- 
cerned, soil  bacteria  are  chiefly  of  importance  in  so  far  as  they 
can  be  washed  out  of  the  soils  into  potable  water  supplies.  An 
important  aspect  of  this  question  thus  is  as  to  the  significance 
of  certain  bacteriological  appearances  in  a  water  in  relation  to 
the  soil  from  which  it  has  come  or  over  which  it  has  flowed. 
In  this  country  (Great  Britain)  these  questions  have  been  chiefly 


BACTERIOLOGICAL   EXAMINATION    OF   SOIL.  129 

investigated  by  Houston,   and  it  is  from  his   papers  that  the 
following  account  is  chiefly  taken. 

Methods  of  Examination.  —  For  examination  of  soil  on  surface  or  not  far 
from  surface,  Houston  recommends  tin  troughs  10  in.  by  3  in.  and  pointed 
at  one  extremity,  to  be  wrapped  in  layers  of  paper  and  sterilised  by  dry  heat. 
If  several  of  these  be  provided,  then  the  soil  can  be  well  rubbed  up  and  a 
sample  secured  and  placed  in  a  sterile  test-tube  for  examination  as  soon  as 
convenient  after  collection.  If  samples  are  to  be  taken  at  some  depth  beneath 
the  surface,  then  a  special  instrument,  of  which  many  varieties  have  been 
devised,  must  be  used.  The  general  form  of  these  is  that  of  a  gigantic  gimlet 
stoutly  made  of  steel.  Just  above  the  point  of  the  instrument  the  shaft  has  in 
it  a  hollow  chamber,  and  a  sliding  lateral  door  in  this  can  be  opened  and  shut 
by  a  mechanism  controlled  at  the  handle.  The  chamber  being  sterilised  and 
closed,  the  instrument  is  bored  to  the  required  depth,  the  door  is  slid  back, 
and  by  varying  devices  it  is  effected  that  the  chamber  is  filled  with  earth  ;  the 
door  is  closed  and  the  instrument  withdrawn. 

In  any  soil  the  two  important  lines  of  inquiry  are  first  as  to  the  total 
number  of  organisms  (usually  reckoned  per  gramme  of  the  fresh  sample),  and 
secondly  as  to  the  varieties  of  organisms  present.  The  number  of  organisms 
present  in  a  soil  is  often,  however,  so  enormous  that  it  is  convenient  to  submit 
only  a  fraction  of  a  gramme  to  examination.  The  method  employed  is  to 
weigh  the  tube  containing  the  soil,  shake  out  an  amount  of  about  the  size  of  a 
bean  into  a  litre  of  distilled  water,  and  re-weigh  the  tube.  The  amount  placed 
in  the  water  is  distributed  as  thoroughly  as  possible  by  shaking,  and,  if  neces- 
sary, by  rubbing  down  with  a  sterile  glass  rod,  and  small  quantities  measured 
from  a  graduated  pipette  are  used  for  the  investigation.  For  estimating  the 
total  number  of  organisms  present  in  the  portion  of  soil  used,  small  quantities, 
say  .1  c.c.  and  i  c.c.,  of  the  fluid  are  added  to  melted  tubes  of  ordinary  alkaline 
peptone  gelatin ;  after  being  shaken,  the  gelatin  is  plated,  incubated  at  22°  C, 
and  the  colonies  are  counted  as  late  as  the  liquefaction,  which  always  occurs 
round  some  of  them,  will  allow.  From  these  numbers  the  total  number  of 
organisms  present  in  the  amount  of  soil  originally  present  can  be  calculated. 

The  numbers  of  bacteria  in  the  soil  vary  very  much.  Accord- 
ing to  Houston's  results,  fewest  occur  in  uncultivated  sandy 
soils,  these  containing  on  an  average  100,000  per  gramme. 
Peaty  soils,  though  rich  in  organic  matter,  also  give  low  results, 
it  being  possible  that  the  acidity  of  such  soils  inhibits  free 
bacterial  growth.  Garden  soils  yield  usually  about  1,500,000 
bacteria  per  gramme,  but  the  greatest  numbers  are  found  in  soils 
which  have  been  polluted  by  sewage,  when  the  figures  may  rise 
to  115,000,000.  In  addition  to  the  enumeration  of  the  numbers 
of  bacteria  present,  it  is  a  question  whether  something  may  not 
be  gained  from  a  knowledge  of  the  number  of  spores  present 
in  a  soil  relative  to  the  total  number  of  bacteria.  This  is  a 


130  BACTERIA   IN   AIR,  SOIL,  AND   WATER. 

point  which  demands  further  inquiry,  especially  by  the  periodic 
investigation  of  examples  of  different  classes  of  soils.  The 
method  is  to  take  I  c.c.  of  such  a  soil  emulsion  as  that  just 
described,  add  it  to  10  c.c.  of  gelatin,  heat  for  ten  minutes  at 
80°  C.  to  destroy  the  non-spored  bacteria,  plate,  incubate,  and 
count  as  before. 

Besides  the  enumeration  of  the  numbers  of  bacteria  present 
in  a  soil,  an  important  question  in  its  bacteriological  examination 
lies  in  inquiring  what  kinds  of  bacteria  are  present  in  any 
particular  case.  Practically  this  resolves  itself  into  studying  the 
most  common  bacteria  present,  for  the  complete  examination  of 
the  bacterial  flora  of  any  one  sample  would  occupy  far  too  much 
time.  Of  these  common  bacteria  the  most  important  are  those 
from  whose  presence  indications  can  be  gathered  of  the  con- 
tamination of  the  soil  by  sewage,  for  from  the  public  health 
standpoint  this  is  by  far  the  most  important  question  on  which 
bacteriology  can  shed  light. 

Bacillus  mycoides. — This  bacillus  is  1.6  to  2-4/x  in  length  and  about  .g/x 
in  breadth.  It  grows  in  long  threads  which  often  show  motility.  It  can  be 
readily  stained  by  such  a  combination  as  carbol-thionin,  and  retains  the  dye 
in  Gram's  method.  All  ordinary  media  will  support  its  growth,  and  in  surface 
growths  on  agar  or  potato  spore  formation  is  readily  produced.  Its  optimum 
temperature  is  about  18°  C.  On  gelatin  plates  it  shows  a  very  characteristic 
appearance.  At  first  under  a  low  power*  it  shows  a  felted  mass  of  filaments 
throwing  out  irregular  shoots  from  the  centre,  and  later  to  the  naked  eye  these 
appear  to  be  in  the  form  of  thick  threads  like  the  growth  of  a  mould.  They 
rapidly  spread  over  the  surface  of  the  medium,  and  the  whole  resembles  a 
piece  of  wet  teased-out  cotton  wool.  The  gelatin  is  liquefied. 

Cladothrices. — Of  these  several  kinds  are  common  in  the  soil.  The 
ordinary  cladothrix  dichotoma  is  among  them.  This  organism  appears  as 
colourless  flocculent  growth  with  an  opaque  centre,  and  can  be  seen  under  the 
microscope  to  send  out  into  the  medium  apparently  branched  threads  which 
vary  in  thickness,  being  sometimes  2  /*.  across.  They  consist  of  rods  enclosed 
in  a  sheath.  These  rods  may  divide  at  any  point,  and  thus  the  terminal  ele- 
ments may  be  pushed  along  the  sheath.  Sometimes  the  sheath  ruptures,  and 
thus  by  the  extrusion  of  these  dividing  cells  and  their  further  division  the 
branching  appearance  is  originated.  Reproduction  takes  place  by  the  forma- 
tion of  gonidia  in  the  interior  of  the  terminal  cells.  These  gonidia  acquire  at 
one  end  a  bundle  of  flagella,  and  for  some  time  swim  free  before  becoming 
attached  and  forming  a  new  colony.  Houston  describes  as  occurring  in  the 
soil  another  variety,  which  with  similar  microscopic  characters  appears  as  a 
brownish  growth  with  a  pitted  surface,  and  diffusing  a  Bismarck-brown  pigment 
into  the  gelatin  which  it  liquefies. 

A  few  experiments  made  with  an  ordinary  field  soil  will,  however,  famil- 


BACTERIOLOGICAL   EXAMINATION    OF   SOIL.  131 

iarise  the  worker  with  the  non-pathogenic  bacteria  usually  present.  We  have 
referred  to  these  two  because  of  their  importance.  In  regard  to  pathogenic 
organisms,  especially  in  relation  to  possible  sewage  contamination,  attention 
is  to  be  directed  to  three  groups  of  organisms,  those  resembling  the  B.  coliy 
the  bacillus  enteritidis  sporogenes,  and  the  streptococcus  pyogenes.  The 
characters  of  the  first  two  of  these  will  be  found  in  the  chapter  on  Typhoid 
Fever;  of  the  third  in  Chapter  VII.  For  the  detection  of  these  bacteria, 
Houston  recommends  the  following  procedure  :  — 

(a)  The  B.  colt  group.  A  third  of  a  gramme  of  soil  is  added  to  10  c.c. 
phenol  broth  (vide  chapter  on  Typhoid  Fever)  and  incubated  at  37°  C.  In 
this  medium  very  few  if  any  other  bacteria  except  those  of  the  B.  coli  group 
will  grow,  so  that  if  after  twenty-four  hours  a  turbidity  appears,  some  of  the 
latter  may  be  suspected  to  be  present.  -  In  such  a  case  a  loopful  of  the  broth 
is  shaken  up  in  5  c.c.  sterile  distilled  water,  and  of  this  one  or  two  loopfuls 
are  spread  over  the  surface  of  a  solid  plate  of  phenol  gelatin  in  a  Petri's  dish 
either  by  means  of  the  loop  or  of  a  small  platinum  spatula,  and  the  plate  is 
incubated  at  20°  C.  Any  colonies  which  resemble  B.  coli  are  then  examined 
by  the  culture  methods  detailed  under  that  organism.  Further,  all  organisms 
having  the  microscopic  appearances  of  B.  coli,  and  which  generally  conform 
to  its  culture  reactions,  are  to  be  reckoned  in  the  coli  group. 

($)  The  Bacillus  enteritidis  sporogenes.  To  search  for  this  organism  one 
gramme  of  the  soil  is  thoroughly  distributed  in  100  c.c.  sterile  distilled  water, 
and  of  this  I  c.c.,  .1  c.c.,  and  .01  c.c.  is  added  to  each  of  three  sterile  milk 
tubes.  These  are  heated  to  80°  C.  for  ten  minutes  and  then  cultivated 
anaerobically  at  37°  C.  for  twenty-four  hours.  If  the  characteristic  appear- 
ances seen  in  such  cultures  of  the  B.  enteritidis  (q.v.}  are  developed,  then  it 
may  fairly  safely  be  deduced  that  it  is  this  organism  which  has  produced  them. 

(c}  The  Streptococcus  pyogenes.  The  method  here  is  to  pour  out  a  tube 
of  agar  into  a  Petri'g  dish,  and  when  it  has  solidified  to  spread  out  .1  c.c.  of 
the  emulsion  of  soil  over  it  and  incubate  at  37°  C.  for  twenty-four  hours.  At 
this  temperature  many  of  the  non-pathogenic  bacteria  grow  with  difficulty,  and 
thus  the  number  of  colonies  which  develop  is  relatively  small.  Colonies  hav- 
ing appearances  resembling  those  of  the  streptococcus  (^.7'.)  can  thus  be 
investigated. 

We  may  now  give  in  brief  the  results  at  which  Houston  has 
arrived  by  the  application  of  these  methods.  First  of  all,  un- 
cultivated soils  contain  very  few,  if  any,  representatives  of  the 
B.  mycoides,  and  this  is  also  true  to  a  less  extent  of  the  clado- 
thrices.  Cultivated  soils,  on  the  other  hand,  do  practically 
always  cpntain  these  organisms.  With  regard  to  the  B.  coli, 
its  presence  in  a  soil  must  be  looked  on  as  indicative  of  recent 
pollution  with  excremental  matter.  The  presence  of  B.  enteri- 
tidis sporogenes  is  also  evidence  of  such  pollution,  but  from  the 
fact  that  it  is  a  sporing  organism  this  pollution  may  not  have  been 
recent.  With  regard' to  the  streptococci,  on  the  other  hand,  the 


.132  BACTERIA   IN   AIR,  SOIL,  AND   WATER. 

•opinion  is  advanced  that  their  presence  is,  on  account  of  their 
want  of  viability  outside  the  animal  body,  to  be  looked  on  as 
evidence  of  extremely  recent  excremental  pollution.  The  very 
•great  importance  of  these  results  in  relation  to  the  bacterio- 
logical examination  of  water  supplies  will  be  at  once  apparent, 
and  will  be  referred  to  again  in  connection  with  the  subject  of 
water. 

While  such  means  have  been  advanced  for  the  obtaining  of 
indirect  evidence  of  excremental  pollution  of  soil,  and  therefore 
of  a  pollution  dangerous  to  health  from  the  possible  presence 
of  pathogenic  organisms  in  excreta,  investigations  have  also 
been  conducted  with  regard  to  the  viability  in  the  soil  of  patho- 
genic bacteria,  especially  of  those  likely  to  be  present  in  excreta, 
namely,  the  typhoid  and  cholera  organisms.  The  solution  of 
this  problem  is  attended  with  difficulty,  as  it  is  not  easy  to  iden- 
tify these  organisms  when  they  are  present  in  such  bacterial 
mixtures  as  naturally  occur  in  the  soil.  Now  there  is  evidence 
that  bacteria  when  growing  together  often  influence  each  other's 
growth  in  an  unfavourable  way,  so  that  it  is  only  by  studying 
the  organisms  in  question  when  growing  in  unsterilised  soils 
that  information  can  be  obtained  as  to  what  occurs  in  nature. 
For  instance,  it  has  been  found  that  the  B.  typhosus,  when 
grown  in  an  organically  polluted  soil  which  has  been  sterilised, 
can  maintain  its  vitality  for  fifteen  weeks,  but  if  the  conditions 
occurring  naturally  be  so  far  imitated  by  growing  it  in  soil  in 
the  presence  of  a  pure  culture  of  one  or  other  certain  soil  bac- 
teria, it  is  found  that  sometimes  the  typhoid  bacillus,  sometimes 
the  soil  bacterium,  in  the  course  of  a  few  weeks,  or  even  in  a 
few  days,  disappears.  Further,  the  character  of  the  soil  exer- 
cises an  important  effect  on  what  happens ;  for  instance,  the 
typhoid  bacillus  soon  dies  out  in  a  virgin  sandy  soil,  even  when 
it  is  the  only  organism  present.  In  experiments  made  by  sow- 
ing cultures  of  cholera  and  diphtheria  in  plots  in  a  field  it  was 
found  that  after,  at  the  longest,  forty  days  they  were  no  longer 
recognisable.  Further,  it  is  a  question  whether  ordinary  disease 
organisms,  even  if  they  remain  alive,  can  multiply  to  any  great 
extent  in  nature.  If  we  are  dealing  with  a  sporing  organism 
such  as  the  B.  anthracis,  the  capacity  for  remaining  in  a  quies- 
cent condition  of  potential  pathogenicity  is,  of  course,  much 
greater. 


BACTERIOLOGICAL   EXAMINATION   OF    SOIL. 


WATER. 


133 


In  the  bacteriological  examination  of  water  three  lines  of  in- 
quiry may  have  to  be  followed.  First,  the  number  of  bacteria 
per  cubic  centimetre  may  be  estimated.  Second,  the  kinds  of 
bacteria  present  may  be  investigated.  Third,  it  may  be  neces- 
sary to  ask  if  a  particular  organism  is  present,  and,  if  so,  in 
what  number  per  cubic  centimetre  it  occurs. 

Methods.  —  All  samples  of  water  taken  for  analysis  should  be,  if  possible, 
treated  at  once  by  one  or  other  of  the  methods  indicated  below.  This  is  espe- 
cially necessary  in  warm  weather,  where  the  delay  even  of  an  hour  or  two  in 
transportation  of  the  sample  to  the  laboratory  will  greatly  increase  the  num- 
ber of  bacteria  present,  and  lead  to  erroneous  ideas  of  the  actual  character  of 
the  water  under  examination.  When  transportation  is 
unavoidable,  the  samples  should  be  packed  in  ice,  and 
sent  forward  with  the  shortest  possible  delay  to  the 
laboratory.  The  collection  of  water  is  usually  made  in 
four-ounce,  wide-mouthed,  stoppered  bottles,  which  are  to 
be  sterilised  by  dry  heat  (the  stopper  must  be  sterilised 
separately  from  the  bottle,  and  not  inserted  in  the  latter 
until  both  are  cold ;  otherwise  it  will  be  so  tightly  held 
as  to  make  removal  very  difficult).  In  using  such  a 
bottle  for  collecting  water  from  rivers,  ponds,  or  lakes 
at  various  depths  it  is  advisable  to  make  use  of  one 
of  the  several  mechanical  devices  commonly  used  for 
such  work  (see  Fig.  60),  in  which  the  bottle  is  secured 
in  a  metal  frame  at  one  end  of  a  graduated  pole,  and 
lowered  into  the  water;  the  stopper  then  is  extracted 
half  its  distance  up  the  neck  of  the  bottle  by  pulling  upon 
a  spring-wire  attachment  which  clutches  the  stopper,  and 
when  the  bottle  is  full  the  wire  being  released  from  the 
finger  carries  back  the  stopper  into  place.  The  operator 
is  warned  not  to  touch  the  water-bed  in  making  a  col- 
lection, nor  surface  scum,  for  such  contain  large  numbers 
of  bacteria,  and  in  the  collection  of  water  from  a  house- 
tap  it  is  advisable  that  the  water  be  let  run  for  at  least 
an  hour  or  two  previous  to  taking  the  sample. 

Upon  arrival  at  the  laboratory,  if  the  samples  had 
to  be  transported,  the  bottles  are  shaken  vigorously  and 
by  means  of  sterile  pipettes  of  i  c.c.  capacity,  divided  into 
tenths,  quantities  of  water  varying  from  ^  to  I  c.c.  are 
transferred  to  melted  tubes  of  gelatin  or  agar,    whose         FIG.  60.  — Appara- 
reaction  is  1.5+   (acid),  and  plated,  and  incubated  at  a   tus  for  collecting  water 
temperature  not  over  22°  C.  nor  under  16°  C.     Where  a   s 
water  is  suspected  to  contain  large  numbers  of  bacteria  it  is  well  to  transfer 
i  c.c.  to  a  small  flask  containing  99  c.c.  of  sterile  water,  and,  shaking  thoroughly, 


134  BACTERIA   OF   AIR,  SOIL,  AND  WATER. 

remove  fractions  of  a  cubic  centimetre  as  before,  and  place  in  the  incubator. 
Upon  the  second,  third,  and  fourth  days  the  colonies  which  have  developed 
should  be  counted  by  means  of  Lafars  counter  (if  Petri's  dishes  have  been 
used)  or  a  Wolffhiigel  apparatus  if  glass  plates  were  employed  (see  Fig.  37). 
These  counters  are  suitable  pieces  of  glass  upon  whose  surfaces  are  ruled 
square  centimetres  and  fractions  thereof,  which  render  errors  in  counting  very 
small  indeed. 

Where  a  plate  contains  comparatively  few  colonies  the  whole  number  of 
squares  should  be  counted,  but  where  a  large  number  of  colonies  are  present 
one  may  count  ten  or  fifteen  representative  squares  at  random  and  determine 
approximately  the  total  number  of  colonies  present.  All  such  counts  are  then 
to  be  properly  reckoned  as  bacteria  per  cubic  centimetre,  allowing  that  each 
colony  has  originated  from  a  single  bacterium  (this  is  not,  however,  strictly 
the  case,  but  where  the  sample  has  been  vigorously  shaken  before  plating,  it  is 
for  all  practical  purposes  sufficiently  near  the  truth). 

Regarding  what  may  be  considered  as  an  impure  water  and  one  to  be  con- 
demned, no  hard  and  fast  statement  can  be  made,  for  under  natural  conditions 
some  waters  contain  a  much  higher  number  of  bacteria  than  do  others.  So  to 
arrive  at  a  proper  basis  for  judging  of  the  conditions  of  purity  or  impurity, 
frequent  repeated  analyses  must  be  made  throughout  a  year  with  especial  refer- 
ence to  species  determination,  more  particularly  as  regards  the  presence  of 
bacillus  coli,  and  the  condition  of  the  watershed  rigidly  examined. 

With  regard  to  the  objects  with  which  the  bacteriological 
examination  of  water  may  be  undertaken,  though  these  may  be 
of  a  purely  scientific  character,  they  usually  aim  at  contributing 
to  the  settlement  of  questions  relating  to  the  potability  of 
waters,  to  their  use  in  commerce,  and  to  the  efficiency  of  pro- 
cesses undertaken  for  the  purification  of  waters  which  have 
undergone  pollution.  The  last  of  these  objects  is  often  closely 
associated  with  the  first  two,  as  the  question  so  often  arises 
whether  a  purification  process  is  so  efficient  as  to  make  the 
water  again  fit  for  use. 

Water  derived  from  any  natural  source  contains  bacteria, 
though,  as  in  the  case  of  some  artesian  wells  and  some  springs, 
the  numbers  may  be  very  small,  e.g.  4  to  100  per  c.c.  In  rain, 
snow,  and  ice  there  are  often  great  numbers,  those  in  the  first 
two  being  derived  from  the  air.  Great  attention  has  been  paid 
to  the  bacterial  content  of  wells  and  rivers.  With  regard  to  the 
former,  precautions  are  necessary  in  arriving  at  a  judgment.  If 
the  water  in  a  well  has  been  standing  for  some  time,  multiplica- 
tion of  bacteria  may  give  a  high  value.  To  meet  this  difficulty, 
if  practicable  the  well  ought  to  be  pumped  dry  and  then  allowed 
to  fill,  in  order  to  get  at  what  is  really  the  important  point, 


FACTORS    INFLUENCING   RESULTS.  135 

namely,  the  bacterial  content  of  the  water  entering  the  well 
Again,  if  the  sediment  of  the  well  has  been  stirred  up  a  high 
value  is  obtained.  Ordinary  wells  of  medium  depth  contain 
from  100  to  2000  per  c.c.  With  regard  to  rivers  very  varied  re- 
sults are  obtained.  In  any  one  river  the  numbers  present  vary 
at  different  seasons  of  the  year,  whilst  the  prevailing  tempera- 
ture, the  presence  or  absence  of  decaying  vegetation,  or  of 
washings  from  land,  and  dilution  with  large  quantities  of  pure 
spring  water,  are  other  important  features.  Thus  the  Frank- 
lands  found  the  rivers  Thames  and  Lea  purest  in  summer,  and 
this  they  attributed  to  the  fact  that  in  this  season  there  is  most 
spring  water  entering,  and  very  little  water  as  washings  off 
land.  In  the  case  of  other  rivers  the  bacteria  have  been  found 
to  be  fewest  in  winter.  Thus  a  great  many  circumstances 
must  be  taken  into  account  in  dealing  with  mere  enumerations 
of  water  bacteria.  Such  enumerations  are  only  useful  when 
they  are  taken  simultaneously  over  a  stretch  of  river,  with 
special  reference  to  the  sources  of  the  water  entering  the  river. 
Thus  it  is  usually  found  that  immediately  below  a  sewage 
effluent  the  bacterial  content  rises,  though  in  a  comparatively 
short  distance  the  numbers  may  decrease  enormously,  and  it 
may  be  that  the  river  as  far  as  numbers  are  concerned  may 
appear  to  return  to  its  previous  bacterial  content.  The  numbers 
of  bacteria  present  in  rivers  vary  so  enormously  that  there  is 
little  use  in  quoting  figures,  most  information  being  obtainable 
by  comparative  enumerations  before  and  after  a  given  event  has 
occurred  to  a  particular  water.  Such  a  method  is  thus  of  great 
use  in  estimating  the  efficacy  of  the  filter  beds  of  a  town  water 
supply.  These  usually  remove  from  95  to  98  per  cent  of  the 
bacteria  present.  Again,  it  is  found  that  the  storage  of  water 
diminishes  the  number  of  bacteria  present.  The  highest  counts 
of  bacteria  per  c.c.  are  observed  with  sewage  ;  for  example,  in  the 
London  sewage  the  numbers  range  from  six  to  twelve  millions. 
Much  more  important  than  the  mere  enumeration  of  the  bac- 
teria present  in  a  water  is  the  question  whether  these  include 
forms  pathogenic  to  man.  The  chief  interest  here,  so  far  as 
Europe  is  concerned,  lies  in  the  fact  that  typhoid  fever  is  so 
frequently  water-borne,  but  cholera  and  certain  other  intestinal 
diseases  have  a  similar  source.  The  search  in  waters  for  the 
organisms  concerned  in  these  diseases  is  a  mattej^j^he  greatest 


136  BACTERIA   IN    AIR,  SOIL,  AND    WATER. 

difficulty,  for  each  belongs  to  a  group  of  organisms  morpho- 
logically similar,  very  widespread  in  nature,  and  many  of  which 
have  little  or  no  pathogenic  action.  The  biological  characters 
of  these  organisms  will  be  given  in  the  chapters  devoted  to  the 
diseases  in  question,  but  here  it  may  be  said  that  from  the 
public  health  standpoint  the  making  of  their  being  found  a 
criterion  for  the  condemning  of  a  water  is  impracticable. 
There  is  no  doubt  that  the  typhoid  and  cholera  bacteria  can 
exist  for  some  time  in  water  —  at  least  this  has  been  found 
to  be  the  case  when  sterile  water  has  been  inoculated  with 
these  bacteria.  But  to  what  extent  the  same  is  true  when  they 
are  placed  in  natural  conditions,  which  involve  their  living  in 
the  presence  of  other  organisms,  is  unknown,  for  it  may  be 
safely  said  that  by  no  known  method  can  the  presence  of  either 
be  demonstrated  in  the  complex  mixtures  which  occur  in  nature. 
With  regard  to  the  typhoid  bacillus,  of  late  the  tendency  has 
been  to  seek  for  the  presence  of  indirect  bacteriological  evi- 
dence which  might  point  in  the  direction  of  the  possibility  of 
the  presence  of  this  organism. 

Methods  of  detecting  B.  coli  in  Water.  —  The  isolation  of  B.  coli  is  brought 
about  in  several  ways.  One  method  aims  at  giving  the  total  number  of  colo- 
nies per  cubic  centimetre  by  plating  various  fractions  of  i  c.c.  in  a  2  per  cent 
lactose  agar,  to  which  neutral  litmus  tincture  has  been  added,  and  incubating 
at  37°  C.  It  is  found  that  a  large  number  of  water  bacteria  will  not  grow  at  this 
temperature,  whereas  B.  coli  grows  well,  its  colonies  becoming  coloured  red  by 
the  action  of  the  bacilli  on  the  lactose  and  thus  are  readily  identified. 

Theobald  Smith  recommends  the  use  of  the  fermentation  tube.  A  series 
of  such  tubes,  containing  i  to  2  per  cent  of  glucose  broth,  are  inoculated  with 
variable  quantities  of  water  and  incubated  for  48  hours  at  37°  C.,  and  those 
showing  25-40  per  cent  of  gas  are  removed  and  plates  made  from  their  con- 
tents and  search  made  for  the  presence  of  B.  coli  by  the  usual  cultural  tests. 

Stone  has  broadened  the  application  of  the  fermentation  method  of  Smith 
by  removing  .5  c.c.  of  the  contents  of  those  tubes  showing  the  proper  quantity 
of  gas,  and  adding  it  to  a  tube  containing  10  c.c.  of  neutral  broth  and  .3  c.c.  of 
Pariettrs  solution,1  and  placing  in  the  thermostat  for  24  hours  at  38°  C.  If 
growth  results,  .5  c.c.  is  removed  to  a  fermentation  tube  and  incubated  as 
before ;  then  if  no  gas  is  formed  it  is  presumed  that  the  fermenting  organism 
met  with  was  not  B.  coli,  but  on  the  other  hand,  if  gas  is  formed  B.  coli  is 
doubtless  present  and  can  readily  be  identified  by  plating  out  and  cultivating 
on  the  various  media. 

1  Parietti's  solution  consists  of          Carbolic  acid          ...       5  grammes. 

Hydrochloric  acid  (pure)       .       4         „ 
Water  (distilled)  .         .  ,       .   100  cc. 


DETECTION    OF    B.  COLI    IN    WATER. 


137 


MacConkey  has  devised  bile-salt  media,  which  he  claims  is  of  great  aid 
in  identifying  B.  coli  and  its  close  relations.  Incubation  must  proceed  at 
42°  C. 


A.    Bile-salt  Agar. 

Agar 1.5  grms. 

Sodium  taurocholate  (pure)  .5  „ 
Peptone  .  .  .  .  2.0  „ 
Water  ....  100.0  c.c. 
This  is  boiled,  clarified,  and 

filtered  as  usual,  then, 
Lactose        .         .         .         .       i.o  grm. 

is  added,  and  the  medium  tubed  and 

sterilised  for  three  successive  days  in  a 

Koch  steriliser. 


B.    Bile-salt  Broth. 

Sodium  taurocholate  (pure)       0.5  grms. 
Peptone       .         .         .         .       2.0      „ 
Glucose        .         .         .  0.5 

Water          ....   100.0  c.c. 
Boil,    filter,    and    add    sufficient    neutral 
litmus;  fill  fermentation  tubes  (Smith's 
or  Durham's)  and  sterilise  as  usual  in 
Koch  steriliser. 


In  the  agar  medium  the  surface  colonies  of  B.  coli  are  found  to  be  large,  round, 
whitish,  with  yellow  centres  and  quite  opaque,  whilst  in  the  medium  surrounding  the 
deep  colonies  is  a  distinct  hazy  halo  around  each,  which  soon  diffuses  throughout  the 
entire  medium,  if  the  deep  colonies  are  at  all  numerous.  The  high  temperature  of 
incubation,  in  combination  with  the  ingredients,  proves  unfavourable  to  develop- 
ment of  bacteria  other  than  intestinal  forms.  Grunbaum  and  Hume  advise  the  addi- 
tion of  I  per  cent  of  a  half  per  cent  watery  solution  of  neutral  red  to  this  medium, 
its  qualities  being  thereby  greatly  enhanced.  Surface  colonies  of  B.  coli,  and  the 
medium  in  their  immediate  locality,  are  coloured  a  bright  violet-red,  whilst  the  typhoid 
colonies  are  yellowish-white,  and  the  medium  surrounding  them  assumes  an  amber 
hue.  This  reaction  is  very  sharp  if  the  medium,  before  sterilisation,  is  standardised 
by  adding  .4  cc.  normal  sodium  hydrate  solution  per  litre,  after  making  neutral  to 
litmus.  Where  the  broth  is  used,  gas-formation  and  cloudiness  betoken  the  presence 
of  B.  coli;  in  absence  of  gas-formation,  cloudiness  may  point  to  the  presence  of 
B.  typhosus  or  B.  fecalis  alkaligenes,  which  can  readily  be  confirmed,  or  not,  by  the 
usual  means  of  identification. 

The  whole  question  turns  on  the  possibility  of  recognising 
bacteriologically  the  contamination  of  water  with  sewage. 
Klein  and  Houston  here  insist  on  the  fact  that  in  crude  sewage 
the  B.  coli  or  the  members  of  the  coli  group  are  practically 
never  fewer  than  100,000  per  c.c.,  and  therefore  if  in  a  water 
this  organism  forms  a  considerable  proportion  of  the  total 
number  of  organisms  present,  then  there  is  grave  reason  for 
suspecting  sewage  pollution.  As,  however,  this  organism  is 
fairly  widespread  in  nature,  they  hold  that  valuable  supporting 
evidence  is  found  in  the  presence  of  the  B.  enteritidis  sporogenes 
and  of  the  streptococci,  both  of  which  are  probably  constant 
inhabitants  of  the  human  intestine.  The  spores  of  the  former 
usually  number  100  per  c.c.  in  sewage,  and  the  presence  of  the 


138  BACTERIA   IN    AIR,  SOIL,  AND   WATER. 

latter  can  always  be  recognised  in  .001  grm.  of  human  faeces. 
The  deductions  to  be  drawn  from  the  presence  of  these  in  water 
are  the  same  as  those  to  be  drawn  from  their  presence  in  soil. 
A  further  point  here  is  that  it  is  well,  wherever  practicable,  that 
the  indirect  evidence  as  to  the  potability  of  a  water  which  is 
usually  derived  from  chemical  analysis  should  be  supplemented 
by  a  bacteriological  search  for  the  three  groups  of  organisms 
mentioned.  It  has  been  found  that  in  water  artificially  polluted 
with  sewage  containing  them,  they  can  be  detected  by  bacterio- 
logical methods  in  mixtures  from  ten  to  a  hundred  times  more 
dilute  than  those  in  which  the  pollution  can  be  detected  by 
purely  chemical  methods. 

Bacterial  Treatment  of  Sewage.  —  Of  late  years  the  opinion 
has  been  growing  that  the  most  appropriate  method  of  dealing 
with  the  disposal  of  sewage  is  to  imitate  as  far  as  possible  the 
processes  which  occur  in  nature  for  the  breaking  up  of  organic 
material.  These  practically  depend  entirely  on  bacterial  action. 
Hence  the  rationale  of  the  most  approved  methods  of  sewage 
disposal  is  to  encourage  the  growth  of  bacteria  which  naturally 
exist  in  sewage,  and  which  are  capable  of  breaking  up  organic 
compounds  and  of  converting  the  nitrogen  into  nitrates  and 
nitrites.  The  technique  by  which  this  is  accomplished  is  very 
varied  and  sometimes  rather  empirical,  but  probably  the  general 
principles  underlying  the  different  methods  are  comparatively 
simple.  It  is  probable  that  for  the  complete  destruction  of  the 
organic  matter  of  sewage  both  aerobic  and  anaerobic  Bacteria 
are  required,  though  on  this  point  there  may  be  some  difference 
of  opinion.  Certainly  very  fair  results  are  obtained  when  ap- 
parently the  conditions  chiefly  favour  aerobic  organisms  alone. 
This  is  usually  effected  by  running  the  sewage  on  to  beds  of 
coke,  allowing  it  to  stand  for  some  hours,  slowly  running  the 
effluent  out  through  the  bottom  of  the  bed,  and  leaving  the  bed 
to  rest  for  some  hours  before  recharging.  The  final  result  is 
better  if  the  effluent  be  afterwards  run  over  another  similar 
coke-bed.  According  to  some  authorities  the  sewage,  as  it  runs 
into  the  first  bed,  takes  up  from  the  air  considerable  free  oxygen, 
which,  however,  soon  disappears  during  the  stationary  period,  so 
that  on  leaving  the  first  bed  the  sewage  contains  little  oxygen. 
In  the  latter  part  of  its  stay  it  has  thus  been  submitted  to  ana- 
erobic conditions.  Further,  while  by  the  passage  of  the  effluent 


BACTERIAL   TREATMENT   OF    SEWAGE.  139 

•out  of  the  first  bed  oxygen  is  sucked  in,  it  rapidly  disappears, 
and  during  the  greater  part  of  the  resting  stage  the  interstices 
of  the  bed  are  filled  with  carbonic  acid  gas,  with  nitrogen  partly 
derived  from  the  air,  partly  from  putrefactive  processes,  and 
thus  in  the  filter  anaerobic  conditions  prevail,  under  which  the 
bacteria  can  act  on  the  deposit  left  on  the  coke.  On  this  latter 
point  there  is  difference  of  opinion,  for,  in  examining  London 
sewage,  Clowes  has  found  oxygen  present  in  abundance  from 
four  to  forty  hours  after  the  sewage  has  been  run  off.  Probably 
the  best  results  in  sewage  treatment  are  obtained  when  it  is 
practicable  to  introduce  a  step  where  there  can  be  no  doubt 
that  the  conditions  are  anaerobic.  This  involves  as  a  prelimi- 
nary stage  the  treatment  of  the  sewage  in  what  is  called  a  septic 
tank,  and  the  method  has  been  adopted  at  Exeter,  Sutton,  and 
Yeovil  in  England,  and  very  fully  worked  out  in  America  by 
the  State  Board  of  Health  of  Massachusetts.  In  the  explana- 
tion given  of  the  rationale  of  this  process,  sewage  is  looked  on 
as  existing  in  three  stages,  (i)  First  of  all,  fresh  sewage  —  the 
newly  mixed  and  very  varied  material  as  it  enters  the  main  sew- 
ers. (2)  Secondly,  stale  sewage  —  the  ordinary  contents  of  the 
main  sewers.  Here  there  is  abundant  oxygen,  and  as  the  sew- 
age flows  along  there  occurs  by  bacterial  action  a  certain  forma- 
tion of  carbon  dioxide  and  ammonia  which  combine  to  form 
ammonium  carbonate.  This  is  the  sewage  as  it  reaches  the 
purification  works.  Here  a  preliminary  mechanical  screening 
may  be  adopted,  after  which  it  is  run  into  an  air-tight  tank 
—  the  septic  tank.  (3)  It  remains  there  for  from  twenty-four 
to  thirty-six  hours,  and  becomes  a  foul-smelling  fluid  —  the 
.septic  sewage.  The  chemical  changes  which  take  place  in  the 
septic  tank  are  of  a  most  complex  nature.  The  sewage  entering 
it  contains  little  free  oxygen,  and  therefore  the  bacteria  in  the 
tank  are  probably  largely  anaerobic,  and  the  changes  which 
they  originate  consist  of  the  formation  of  comparatively  simple 
compounds  of  hydrogen  with  carbon,  sulphur,  and  phosphorus. 
As  a  result  there  is  a  great  reduction  in  the  amount  of  organic 
nitrogen,  of  albuminoid  ammonia,  and  of  carbonaceous  matter. 
The  latter  fact  is  important,  as  the  clogging  of  ordinary  filter 
beds  is  largely  due  to  the  accumulation  of  such  material,  and  of 
matters  generally  consisting  of  cellulose.  One  further  impor- 
tant effect  is  that  the  size  of  the  deposited  matter  is  decreased, 


140  BACTERIA   IN   AIR,  SOIL,  AND   WATER. 

and  therefore  it  is  more  easily  broken  up  in  the  next  stage  of 
the  process.  This  consists  of  running  the  effluent  from  the 
septic  tank  on  to  filter  beds,  preferably  of  coke,  where  a  further 
purification  process  takes  place.  By  this  method  there  is  first 
an  anaerobic  treatment  succeeded  by  an  aerobic ;  in  the  latter 
the  process  of  nitrification  occurs  by  means  of  the  special  bac- 
teria concerned.  The  results  are  of  a  most  satisfactory  nature. 
Sometimes  the  effluent  from  a  sewage  purification  system 
contains  as  many  bacteria  as  the  sewage  entering,  but,  especially 
by  means  of  the  septic  tank  method,  there  is  often  a  marked 
diminution.  It  is  said  by  some  that  pathogenic  bacteria  do  not 
live  in  sewage,  but  in  an  effluent  B.  coli,  B.  enteritidis,  and 
streptococci  have  been  constantly  found,  so  that  the  observation 
is  of  little  value,  and  it  is  only  by  great  dilution  and  prolonged 
exposure  to  the  conditions  present  in  running  water  that  such 
an  effluent  can  be  again  a  part  of  a  potable  water. 

ANTISEPTICS. 

The  death  of  bacteria  is  judged  of  by  the  fact  that  when  they 
are  placed  on  a  suitable  food  medium  no  development  takes 
place.  Microscopically  it  would  be  observed  that  division  no 
longer  occurred,  and  that  in  the  case  of  motile  species  move- 
ment would  have  ceased,  but  such  an  observation  has  only 
scientific  interest.  From  the  importance  of  being  able  to  kill 
bacteria  an  enormous  amount  of  work  has  been  done  in  the  way 
of  investigating  the  means  of  doing  so  by  chemical  means,  and 
the  bodies  having  such  a  capacity  are  called  antiseptics.  It  is 
now  known  that  the  activity  of  these  agents  is  limited  to  the 
killing  of  bacteria  outside  the  animal  body,  but  still  even  this  is 
of  high  importance. 

Methods.  —  These  vary  very  much.  In  early  inquiries  a  great  point  was 
made  of  the  prevention  of  putrefaction,  and  work  was  done  in  the  way  of 
finding  how  much  of  an  agent  must  be  added  to  a  given  solution  such  as  beef 
infusion,  urine,  etc.,  in  order  that  the  bacteria  accidentally  present  might  not 
develop ;  but  as  bacteria  vary  in  their  powers  of  resistance,  the  method  was 
unsatisfactory,  and  now  an  antiseptic  is  usually  judged  of  by  its  effects  on  pure 
cultures  of  definite  pathogenic  microbes,  and  in  the  case  of  a  sporing  bacterium 
the  effect  on  both  the  vegetative  and  spore  forms  is  investigated.  The  organ- 
isms most  used  are  the  staphylococcus  pyogenes,  streptococcus  pyogenes, 
and  the  organisms  of  typhoid,  cholera,  diphtheria,  and  anthrax  —  the  latter 
being  most  used  for  testing  the  action  on  spores.  The  best  method  to  employ 


THE   ACTION    OF   ANTISEPTICS.  .  141 

is  to  take  sloped  agar  cultures  of  the  test  organism,  scrape  off  the  growth, 
and  mix  it  up  with  a  small  amount  of  distilled  water  and  filter  this  emulsion 
through  a  plug  of  sterile  glass  wool  held  in  a  small  sterile  glass  funnel,  add  a 
measured  quantity  of  this  fluid  to  a  given  quantity  of  a  solution  of  the  anti- 
septic in  distilled  water,  then  after  the  lapse  of  the  period  of  observation  to 
remove  one  or  two  loopfuls  of  the  mixture  and  place  them  in  a  great  excess  of 
culture  medium.  Here  it  is  preferable  to  use  fluid  agar,  which  is  then  plated  and 
incubated ;  such  a  procedure  is  preferable  to  the  use  of  bouillon  tubes,  as  any 
colonies  developing  can  easily  be  recognised  as  belonging  to  the  species  of 
bacterium  used.  In  dealing  with  strong  solutions  of  chemical  agents  it  is 
necessary  to  be  sure  that  the  culture  fluid  is  in  great  excess,  so  that  the  small 
amount  of  the  antiseptic  which  is  transferred  with  the  bacteria  may  be  diluted 
far  beyond  the  strength  at  which  it  still  can  have  any  noxious  influence. 
Sometimes  it  is  possible  at  the  end  of  the  period  of  observation  to  change 
the  antiseptic  into  inert  bodies  by  the  addition  of  some  other  substance  and 
then  test  the  condition  of  the  bacteria,  and  if  the  inert  substances  are  fluid 
there  is  no  objection  to  this  proceeding,  but  if  in  the  process  a  precipitate 
results,  then  it  is  better  not  to  have  recourse  to  such  a  method,  as  sometimes 
the  bacteria  are  carried  down  with  the  precipitate  and  may  escape  the  culture 
test.  The  advisability  of,  when  possible,  thus  chemically  changing  the  antiseptic 
was  first  brought  to  notice  by  the  criticism  of  Koch's  statements  as  to  the  efficacy 
of  mercuric  chloride  in  killing  the  spores  of  the  B.  anthracis.  The  method 
he  employed  in  his  experiments  was  to  soak  silk  threads  in  an  emulsion  of 
anthrax  spores  and  dry  them.  These  were  then  subjected  to  the  action  of 
the  antiseptic,  well  washed  in  water,  and  laid  on  the  surface  of  agar.  It  was 
found,  however,  that  with  threads  exposed  to  a  far  higher  concentration  of 
the  corrosive  sublimate  than  Koch  had  stated  was  sufficient  to  prevent  growth, 
if  the  salt  were  broken  up  by  the  action  of  ammonium  sulphide  and  this 
washed  off,  growth  of  anthrax  still  occurred  when  the  threads  were  laid  on  agar. 
The  explanation  given  was  that  the  antiseptic  had  formed  an  albuminate  with 
the  case  of  each  spore,  and  that  this  prevented  the  antiseptic  from  acting 
upon  the  contained  protoplasm.  Such  an  occurrence  only  takes  place  with 
spores,  and  the  method  given  above,  in  which  the  small  amount  of  antiseptic 
adhering  to  the  bacteria  is  swamped  in  an  excess  of  culture  fluid,  can  safely  be 
followed,  especially  when  a  series  of  antiseptics  is  being  compared. 

The  Action  of  Antiseptics.  —  In  inquiries  into  the  actions  of 
antiseptics  attention  to  a  great  variety  of  factors  is  necessary, 
especially  when  the  object  is  not  to  compare  different  antiseptics 
with  one  another,  but  when  the  absolute  value  of  any  body  is 
being  investigated.  Thus  the  medium  in  which  the  bacteria  to 
be  killed  are  situated  is  important;  the  more  albuminous  the 
surroundings  are,  the  greater  degree  of  concentration  is  required. 
Again,  the  higher  the  temperature  at  which  the  action  is  to  take 
place,  the  more  dilute  may  the  antiseptic  be,  or  the  shorter  the 
exposure  necessary  for  a  given  effect  to  take  place.  The  most 


142  BACTERIA   IN    AIR,  SOIL,  AND   WATER. 

important  factor,  however,  to  be  considered  is  the  chemical 
nature  of  the  substances  employed.  Though  nearly  every  sub- 
stance which  is  not  a  food  to  the  animal  or  vegetable  body  is 
more  or  less  harmful  to  bacterial  life,  yet  certain  bodies  have  a 
more  marked  action  than  others.  Thus  it  may  be  said  that  the 
most  important  antiseptics  are  the  salts  of  the  heavy  metals, 
certain  acids,  especially  mineral  acids,  certain  oxidising  and 
reducing  agents,  a  great  variety  of  substances  belonging  to 
the  aromatic  series,  and  volatile  oils  generally.  In  comparing 
different  bodies  belonging  to  any  one  of  these  groups  the  chemi- 
cal composition  or  constitution  is  very  important,  and  if  such 
comparisons  are  to  be  made,  the  solutions  compared  must  be 
equimolecular ;  in  other  words,  the  action  of  a  molecule  of  one 
body  must  be  compared  with  the  action  of  a  molecule  of  another 
body.  This  can  be  done  by  dissolving  the  molecular  weight  in 
grammes  in  say  a  litre  of  water  (see  p.  36).  When  this  is  done 
important  facts  emerge.  Thus,  generally  speaking,  the  com- 
pounds of  a  metal  of  high  atomic  weight  are  more  powerful 
antiseptics  than  those  of  one  belonging  to  the  same  series,  but 
of  a  lower  atomic  weight.  Among  organic  bodies  again  sub- 
stances with  high  molecular  weight  are  more  powerful  than 
those  of  low  molecular  weight  —  thus  butyric  alcohol  is  more 
powerful  than  ethylic  alcohol  —  and  important  differences  among 
the  aromatic  bodies  are  associated  with  their  chemical  consti- 
tution. Thus  among  the  cresols  the  ortho-  and  para-  bodies 
resemble  each  other  in  general  chemical  properties,  and  stand 
apart  from  metacresol ;  they  also  are  similar  in  antiseptic  action, 
and  are  much  stronger  than  the  meta-  body.  The  same  may  be 
observed  in  the  other  groups  of  ortho-,  meta-,  and  para-  bodies. 
Again,  such  a  property  as  acidity  is  important  in  the  action  of  a 
substance,  and,  generally  speaking,  the  greater  the  avidity  of  an 
acid  to  combine  with  an  alkali,  the  more  powerful  an  antiseptic 
it  is.  With  regard  to  oxidising  agents  and  reducing  agents, 
probably  the  possession  of  such  properties  has  been  overrated 
as  increasing  bactericidal  potency.  Thus  in  the  case  of  such 
reducers  as  sulphurous  acid  and  formic  acid,  the  effect  is  appar- 
ently chiefly  due  to  the  fact  that  these  substances  are  acids. 
Formic  acid  is  much  more  efficient  than  formate  of  sodium.  In 
the  case  of  permanganate  of  potassium,  which  is  usually  taken 
as  the  type  of  oxidising  agents  in  this  connection,  it  can  be 


THE   ACTIONS   OF   CERTAIN    ANTISEPTICS.  143 

shown  that  the  greater  amount  of  the  oxidation  which  takes 
place  when  this  agent  is  brought  into  contact  with  bacteria 
occurs  after  the  organisms  are  killed.  Such  an  observation  is, 
however,  not  conclusive  as  to  the  non-efficiency  of  the  oxidation 
process,  for  the  death  of  the  bacteria  might  be  due  to  the  oxida- 
tion of  a  very  small  part  of  the  bacterial  protoplasm.  Apart 
from  the  chemical  nature  of  antiseptic  agents,  the  physical  fac- 
tors concerned  in  their  solution,  especially  when  they  are  elec- 
trolytes, probably  play  a  part  in  their  action.  The  part  played 
by  such  factors  is  exemplified  in  the  important  fact  that  a  strong 
solution  acting  for  a  short  time  will  have  the  same  effect  as  a 
weaker  solution  acting  for  a  longer  time.  From  what  has  been 
said  it  will  be  realised  that  the  real  causes  of  a  material  being 
an  antiseptic  are  very  obscure,  and  at  present  we  can  only  have 
a  remote  idea  of  the  factors  at  work. 

The  Actions  of  Certain  Antiseptics.  —  Here  we  can  only  briefly 
indicate  certain  results  obtained  with  the  more  common  members 
of  the  group. 

Chlorine.  —  All  the  halogens  have  been  found  to  be  power- 
ful antiseptics,  but  from  the  cheapness  with  which  it  can  be  pro- 
duced chlorine  has  been  most  used ;  not  only  is  it  the  chief 
active  agent  in  the  somewhat  complex  action  of  bleaching  pow- 
der, but  it  is  also  the  chief  constituent  of  several  proprietary 
substances,  of  which  "  Electrozone  "  is  a  good  example.  This 
last  substance  is  made  from  electrolysing  sea  water,  when  mag- 
nesia and  chlorine  being  liberated,  magnesium  hypochlorite  and 
magnesium  chloride  are  formed.  In  the  action  of  this  substance 
free  hypochlorous  acid  is  formed,  and  the  effect  produced  is 
thus  similar  to  that  of  bleaching  powder.  Nissen,  investigating 
the  action  of  the  latter,  found  that  i|  per  cent  killed  typhoid 
bacilli  in  faeces  ;  and  Rideal  found  that  I  part  to  400-500  disin- 
fected sewage  in  fourteen  minutes,  and  Delepine's  results  show 
that  i  part  to  50  (equal  to  .66  per  cent  of  chlorine)  rapidly  kills 
the  tubercle' bacillus,  and  I  part  to  10  (equal  to  3.3  per  cent) 
killed  anthrax  spores.  Klein  found  that  .05  per  cent  of  chlorine 
killed  most  bacterial  spores  in  five  minutes. 

Iodine  Terchloride.  —  This  is  a  very  unstable  compound  of 
iodine  and  chlorine,  and  though  it  has  been  much  used  as  an 
antiseptic,  seeing  that  the  substance  only  remains  as  IC13  in  an 
atmosphere  of  chlorine  gas,  it  is  open  to  doubt  whether  the 


144  BACTERIA   IN   AIR,  SOIL,  AND   WATER. 

effects  described  are  not  due  to  a  very  complicated  action  of  free 
hydrochloric  acid,  hydriodic  acid,  and  of  oxyacids  of  chlorine 
and  iodine  produced  by  its  decomposition.  It  is  stated,  how- 
ever, that  the  action  is  very  potent :  a  I  per  cent  solution  is 
said  instantly  to  kill  even  anthrax  spores,  but  if  the  spores  be  in 
bouillon,  death  occurs  after  from  ten  to  twelve  minutes.  In 
serum  the  necessary  exposure  is  from  thirty  to  forty  minutes. 
A  solution  of  i-iooo  will  kill  the  typhoid,  cholera,  and  diphtheria 
organisms  in  five  minutes. 

Nascent  Oxygen.  —  This  is  chiefly  available  in  two  ways  - 
firstly,  when  in  the  breaking  up  of  ozone  the  free  third  atom  of 
the  ozone  molecule  is  seeking  to  unite  with  another  similar  atom  ; 
secondly,  when  peroxide  of  hydrogen  is  broken  up  into  water 
and  an  oxygen  atom  is  thereby  liberated.  In  commerce  the 
activity  of  "  Sanitas "  compounds  is  due  to  the  formation  of 
ozone  by  the  slow  oxidation  of  the  resin,  camphor,  and  thymol 
they  contain. 

Perchloride  of  Mercury.  — Of  all  the  salts  of  the  heavy  met- 
als this  has  been  most  widely  employed,  and  must  be  regarded 
as  one  of  the  most  powerful  and  useful  of  known  antiseptics. 
In  testing  this  action  on  anthrax  spores  there  is  no  doubt  that  in 
the  earlier  results  its  potency  was  overrated  from  a  neglect  of 
the  fact  already  alluded  to,  that  in  the  spore-case  an  albumin  ate 
of  mercury  was  formed  which  prevented  the  contained  proto- 
plasm from  developing,  while  not  depriving  it  of  life.  It  has 
been  found,  however,  that  this  salt  in  a  strength  of  i-ioo  will 
kill  the  spores  in  twenty  minutes,  although  an  hour's  exposure 
to  i-iooo  has  no  effect.  The  best  results  are  obtained  by  the 
addition  to  the  corrosive  sublimate  solution  of  .5  per  cent  of  sul- 
phuric acid  or  hydrochloric  acid ;  the  spores  will  then  be  killed 
by  a  seventy-minute  exposure  to  a  1-200  solution.  When,  how- 
ever, organisms  in  the  vegetative  condition  are  being  dealt  with, 
much  weaker  solutions  are  sufficient ;  thus  anthrax  bacilli  in 
blood  will  be  killed  in  a  few  minutes  by  1-2000,  in  bouillon  by 
1-40,000,  and  in  water  by  1-500,000.  Plague  bacilli  are  killed  by 
one  to  two  minutes'  exposure  to  1-3000.  Generally  speaking,  it 
may  be  said  that  a  1-2000  solution  must  be  used  for  the  practically 
instantaneous  killing  of  vegetative  organisms. 

Perchloride  of  mercury  is  one  of  the  substances  which  have 
been  used  for  disinfecting  rooms  by  distributing  it  from  a  spray 


THE   ACTIONS   OF   CERTAIN   ANTISEPTICS.  145 

producer,  of  which  the  "  Equifex  "  may  be  taken  as  a  type.  With 
such  a  machine  it  is  calculated  that  I  oz.  of  perchloride  of  mer- 
cury used  in  a  solution  of  i-iooo  will  probably  disinfect  3000 
square  feet  of  surface.  Such  a  procedure  has  been  extensively 
used  in  the  disinfection  of  plague  houses,  but  the  use  of  a 
stronger  solution  (1-500  acidulated)  is  probably  preferable. 

Formalin  as  a  commercial  article  is  a  40  per  cent  solution  of 
formaldehyde  in  water.  This  is  a  substance  which  of  late  years 
has  come  much  into  vogue,  and  it  is  undoubtedly  a  valuable 
antiseptic.  A  disadvantage,  however,  to  its  use  is  that,  when 
diluted  and  exposed  to  air,  amongst  other  changes  which  it  under- 
goes it  may  be  transformed,  under  little  understood  conditions, 
into  trioxymethylene  and  paraformaldehyde,  these  being  poly- 
mers of  formaldehyde.  The  bactericidal  values  of  these  mix- 
tures are  thus  indefinite.  Formalin  may  be  used  either  by 
applying  it  in  its  liquid  form  or  as  a  spray,  or  the  gas  which 
evaporates  at  ordinary  temperatures  from  the  solution  may  be 
utilised.  To  disinfect  such  an  organic  mixture  as  pus  contain- 
ing pyogenic  organisms  a  10  percent  solution  acting  for  half  an 
hour  is  necessary.  In  the  case  of  pure  cultures,  a  5  per  cent 
solution  will  kill  the  cholera  organism  in  three  minutes,  anthrax 
bacilli  in  a  quarter  of  an  hour,  and  the  spores  in  five  hours. 
When  such  organisms  as  pyogenic  cocci,  cholera  spirillum,  and 
anthrax  bacillus  infect  clothing,  an  exposure  to  the  full  strength 
of  formalin  for  two  hours  is  necessary,  and  in  the  case  of  anthrax 
spores,  for  twenty-four  hours.  Silk  threads  impregnated  with 
the  plague  bacillus  were  found  to  be  sterile  after  two  minutes' 
exposure  to  formalin. 

The  action  of  formalin  vapour  has  been  much  studied,  as  its 
use  constitutes  a  cheap  method  of  treating  infected  rooms,  in 
which  case  some  spray-producing  machine  is  employed.  It  is 
stated  that  a  mixture  of  8  c.c.  of  formalin  with  40  c.c.  of  water 
is  sufficient  when  vaporised  to  disinfect  I  c.m.,  so  far  as  non- 
sporing  organisms  are  concerned.  It  is  stated  that  I  part 
formalin  in  10,000  of  air  will  kill  the  cholera  vibrio  in  one  hour, 
diphtheria  bacillus  in  three  hours,  the  staphylococcus  pyogenes 
in  six  hours,  and  anthrax  spores  in  thirteen  hours.  In  the  case 
of  organisms  which  have  become  dry  it  is  probable,  however, 
that  much  longer  exposures  are  necessary,  but  on  this  point  we 
have  not  definite  information. 


I46  BACTERIA   IN    AIR,  SOIL,  AND  WATER. 

Formalin  gas  has  only  a  limited  application  ;  it  has  little 
effect  on  dry  organisms,  and  in  the  case  of  wet  organisms  must, 
in  order  to  be  effective,  probably  become  dissolved  so  as  to  give 
the  moisture  a  proportion  analogous  to  the  strengths  given  above 
with  regard  to  the  vapour. 

Sulphurous  Acid.  —  This  substance  has  long  been  in  use, 
largely  from  the  cheapness  with  which  it  can  be  produced  by 
burning  sulphur  in  the  air.  An  atmosphere  containing  98  per 
cent  will  kill  the  pyogenic  cocci  in  two  minutes  if  they  are  wet, 
and  in  twenty  minutes  if  they  are  dry  ;  and  anthrax  bacilli  are 
killed  by  thirty  minutes'  exposure,  but  to  kill  anthrax  spores  an 
exposure  of  one  to  two  hours  to  an  atmosphere  containing  1 1 
per  cent  is  necessary.  For  a  small  room  the  burning  of  about 
a  pound  and  a  half  (most  easily  accomplished  by  moistening  the 
sulphur  with  methylated  spirit)  is  usually  considered  sufficient. 
It  has  been  found  that  if  bacteria  are  protected,  as  by  being 
in  the  middle  of  small  bundles  of  clothes,  no  effect  is  produced 
even  by  an  atmosphere  containing  a  large  proportion  of  the 
sulphurous  acid  gas.  The  practical  applications  of  this  agent 
are  therefore  limited. 

Potassium  Permanganate.  —  The  action  of  this  agent  very 
much  depends  on  whether  it  can  obtain  free  access  to  the  bac- 
teria to  be  killed  or  whether  these  are  present  in  a  solution  con- 
taining much  organic  matter.  In  the  latter  case  the  oxidation 
of  the  organic  material  throws  so  much  of  the  salt  out  of  action 
that  there  may  be  little  left  to  attack  the  organisms.  Koch 
found  that  to  kill  anthrax  spores  a  5  per  cent  solution  required 
to  act  for  about  a  day ;  for  most  organisms  a  similar  solution 
acting  for  shorter  periods  has  been  found  sufficient,  and  in  the 
case  of  the  pyogenic  cocci  a  i  per  cent  solution  will  kill  in  ten 
minutes.  There  is  little  doubt  that  such  weaker  solutions  are  of 
value  in  disinfecting  the  throat  on  account  of  their  non-irritating 
properties,  and  good  results  in  this  connection  have  been  obtained 
in  cases  of  diphtheria.  A  solution  of  I  in  10,000  has  been  found 
to  kill  plague  bacilli  in  five  minutes. 

Carbolic  Acid.  —  Of  all  the  aromatic  series  this  is  the  most 
extensively  employed  antiseptic.  All  ordinary  bacteria  in  the 
vegetative  condition,  and  of  these  the  staphylococcus  pyogenes 
is  the  most  resistant,  are  killed  in  less  than  five  minutes  by  a 
a  to  3  per  cent  solution  in  water,  so  that  the  5  per  cent  solution 


THE   ACTIONS    OF   CERTAIN   ANTISEPTICS.  147 

usually  employed  in  surgery  leaves  a  margin  of  safety.  But  for 
the  killing  of  such  organisms  as  anthrax  spores  a  very  much 
longer  exposure  is  necessary  ;  thus  Koch  found  it  necessary  to 
expose  these  spores  for  four  days  to  ensure  disinfection.  The 
risk  of  such  spores  being  present  in  ordinary  surgical  procedure 
may  be  overlooked,  but  there  might  be  risk  of  tetanus  spores 
not  being  killed,  as  these  will  withstand  fifteen  hours'  exposure 
to  a  5  per  cent  solution. 

In  the  products  of  the  distillation  of  coal  there  occur,  besides 
carbolic  acid,  many  bodies  of  a  similar  chemical  constitution, 
and  many  mixtures  of  these  are  in  the  market  —  the  chief 
being  creolin,  izal,  and  lysol,  all  of  which  are  agents  of  value. 
Of  these  lysol  is  perhaps  the  most  noticeable,  as  from  its 
nature  it  acts  as  a  soap  and  thus  can  remove  fat  and  dirt  from 
the  hands.  A  one-third  per  cent  solution  is  said  to  destroy  the 
typhoid  and  cholera  organisms  in  twenty  minutes.  A  i  per 
cent  solution  is  sufficient  for  ordinary  surgical  procedures. 

lodoform.  — This  is  an  agent  regarding  the  efficacy  of  which 
there  has  been  much  dispute.  There  is  little  doubt  that  it  owes 
its  efficiency  to  its  capacity  for  being  broken  up  by  bacterial 
action  in  such  a  way  as  to  set  free  iodine,  which  acts  as  a  power- 
ful disinfectant.  The  substance  is  therefore  of  value  in  the 
treatment  of  foul  wounds,  such  as  those  of  the  mouth  and  rec- 
tum, where  reducing  bacteria  are  abundantly  present  It  acts 
more  slightly  where  there  are  only  pyogenic  cocci,  and  it  seems 
to  have  a  specially  beneficial  effect  in  tubercular  affections.  In 
certain  cases  its  action  may  apparently  be  aided  by  the  presence 
of  the  products  of  tissue  degeneration. 

From  the  results  which  have  been  given  it  will  easily  be  rec- 
ognised that  the  choice  of  an  antiseptic  and  the  precise  man- 
ner in  which  it  is  to  be  employed  depend  entirely  on  the 
environment  of  the  bacteria  which  are  to  be  killed.  In  many 
cases  it  will  be  quite  impossible,  without  original  inquiry,  to  say 
what  course  is  likely  to  be  attended  with  most  success. 


CHAPTER   V. 
FUNGI:    NON-PATHOGENIC   AND    PATHOGENIC. 

IT  is  quite  outside  the  scope  of  the  present  volume  to  de- 
scribe any  bacteria  other  than  those  giving  rise  to  disease  pro- 
cesses. In  the  course  of  his  work  the  bacteriologist  frequently 
meets  with  ordinary  saprophytic  organisms.  These  may  occur 
in  diseased  organs  in  which  putrefaction  has  already  begun  to 
take  place,  and  they  may  therefore  appear  in  cultures  made 
from  such  organs.  Their  source  in  cultures  may,  further,  be  by 
contamination  from  the  air,  or  from  the  use  of  insufficiently 
sterilised  vessels  or  instruments.  The  positive  characters  of 
the  pathogenic  bacteria  will  be  given,  and  from  these  other 
bacteria  must  be  distinguished  by  the  application  of  the  methods 
of  diagnosis  already  detailed,  or  by  the  special  methods  still  to 
be  described.  There  occur,  however,  from  time  to  time  as  con- 
taminations of  bacterial  cultures,  organisms  of  a  more  compli- 
cated structure  than  the  bacteria,  namely  fungi,  and  therefore 
we  shall  describe  a  few  of  the  typical  forms  of  these. 

The  fungi  have  probably  descended  from  the  algae,  or  both 
have  had  a  common  ancestor.  This  is  shown  by  the  close 
resemblances  in  structure  and  development  which  the  two 
groups  present  to  each  other.  The  chief  differences  centre 
round  the  degeneration  of  structure  which  the  adoption  of 
parasitism  and  saprophytism  entails  on  the  fungi.  In  the 
algae,  reproduction  takes  place  in  both  sexual  and  non-sexual 
ways.  In  the  former  case,  certain  cells  called  gametes  are  set 
apart,  and  by  the  union  of  two  of  these  —  the  embryonic  male 
and  female  elements  —  a  new  cell  called  a  zygospore  is  formed 
which  after  a  period  of  rest  grows  into  a  new  individual.  Some- 
times there  is  a  more  definite  male  element,  the  antherozoid,  and 
a  female  element,  the  oosphere,  and  the  coalescence  of  these 
forms  an  oospore  which  subsequently  behaves  like  a  zygospore. 
In  the  non-sexual  reproduction  there  are  formed  certain  cells 

148 


MUCORIN^   AND    ASCOMYCET^E.  149 

called  sporangia,  the  protoplasm  of  which,  without  being  rein- 
forced from  that  of  another  cell,  proceeds  to  break  up  into 
the  elements  of  new  individuals,  often  called,  when  motile, 
swarm-spores.  Both  forms  of  reproduction  are  usually  mani- 
fested by  each  species.  The  degradation  of  the  fungi  consists 
in  the  gradual  loss  of  the  faculty  of  sexual  reproduction,  so  that, 
in  the  most  extreme  species  of  the  group,  it  does  not  appear  at 
all  and  only  asexual  reproduction  can  be  traced.  We  shall  now 
describe  a  few  of  the  typical  forms  of  these  lower  fungi  which 
are  often  met  with  in  bacteriological  work. 

Mucorinae:  Mucor  Mucedo.  — This  form  occurs  especially  in  the 
putrefaction  of  horse  dung  and  also  in  other  putrefactions.  To 
the  naked  eye  it  appears  as  a  white  or  brownish-white  mass  of 
fine  filaments,  from  which,  here  and  there,  rise  special  filaments 
often  several  inches  long,  having  at  their  terminations  spherical 
brown  swellings,  the  reproductive  elements.  Microscopically, 
the  plant  consists  of  branching  non-septate  filaments.  Such  a 
structure  is  called  a  mycelium.  The  non-sexual  is  the  commonest 
form  of  reproduction  (vide  Fig.  61,  A4).  One  of  the  filaments 
grows  out,  at  its  termination  a  septum  forms,  and  a  globular 
swelling  (the  sporangium)  appears.  This  sporangium  possesses 
a  definite  membrane.  Within  it  from  the  septum  grows  a  club- 
shaped  mass  of  protoplasm  called  the  columella,  to  which  are 
attached  the  swarm-spores  formed  from  the  breaking  up  of  the 
rest  of  the  protoplasm.  When  ripe  the  brood  cell  bursts,  the 
brown  swarm-spores  are  cast  off,  and  from  each  a  new  individual 
arises.  Under  certain  circumstances  sexual  reproduction  occurs 
(vide  Fig.  61,  A 1-3).  Two  filaments  approach  each  other,  and  a 
small  piece  of  the  protoplasm  of  each  being  cut  off  by  a  septum, 
these  parts  coalesce.  A  zygospore  is  thus  formed  from  which 
a  new  filamentous  individual  arises. 

Ascomycetae :  Oidium  Lactis  (Fig.  61,  B).  —  This  is  a  common 
organism  in  sour  milk  and  sour  bread.  It  can  easily  be  culti- 
vated on  gelatin  where  the  colonies  appear  to  consist  of  fine 
white  filaments  radiating  from  a  centre.  Microscopically  here 
and  there  the  filaments  (which  may  be  branched)  are  broken 
up,  especially  at  the  ends,  into  short,  rod-shaped  or  oval  seg- 
ments, often  referred  to  as  the  oidia.  These  behave  like  spores. 
Non-sexual  reproduction  also  takes  place  by  the  formation,  within 
certain  special  sporangia  in  the  filament  called  asci,  of  a  definite 


150         FUNGI:    NON-PATHOGENIC   AND   PATHOGENIC. 

number  of  spores,  to  which  the  special  name  of  ascospores  is 
applied. 

Perisporiaceae:    (i)   Aspergillus  Niger  (vide  Fig..  61,  C).  - 
This,  with  other  varieties  of  the    same  group  is*  of    frequent 


FlG.  61. — A.  Mucor  mucedo;  (i),  (2),  (3)  stages  in  formation  of  a  zygospore;  (4)  a 
sporangium  containing  spores.  B.  Oidium  lactis.  C.  Aspergillus  glaucus  (De  Bary)  ; 
(i)  mycelium;  (2)  and  (5)  gonidiophore  bearing  spores ;  (3),  (4)  a  perithecium  (4  con- 
tains rudimentary  asci)  ;  (6)  piece  of  gonidiophore;  (a)  sterigma;  (b}  spore.  D. 
Branched  gonidiophore  of  penicillium  glaucum  bearing  spores.  E,  F.  Saccharomyces 
cerevisise,  cells  are  budding.  G.  Ditto,  formation  of  endospores  (after  Hansen). 


PERISPORIACE^E.  151 

occurrence,  especially  in  vegetable  putrefactions.  It  grows 
readily  in  gelatin.  It  consists,  to  the  naked  eye,  like  the  other 
fungi  described,  of  a  mass  of  felted  filaments  which  microscop- 
ically are  seen  to  form  a  septate  branching  mycelium.  Though 
it  is  a  matter  of  doubt  whether  sexual  reproduction  takes  place, 
two  forms  of  reproduction  occur,  the  variety  depending  largely 
on  the  nutrition  of  the  plant.  The  less  common  form  is  effected 
by  means  of  the  formation  of  structures  known  as  perithecia, 
and  it  may  perhaps  be  that  the  perithecia  owe  their  formation 
to  a  sexual  act.  From  a  mycelial  branch  there  arises  a  filament 
(or  hypha)  which  becomes  specially  coiled  and  transversely  sep- 
tate at  its  end.  From  the  base  of  the  lowest  coil  of  the  spiral 
two  or  three  hyphae  grow  up  towards  its  apex.  One  of  these, 
being  the  first  to  reach  the  apex,  was  regarded  by  De  Bary  as 
a  male  organ.  The  others,  by  branching  copiously,  produce  a 
mass  of  closely  woven  hyphae,  which  form  a  closed  wall  to  this 
structure,  which  is  the  perithecium  referred  to.  Within  it  nu- 
merous asci  arise  as  the  ultimate  ramifications  of  branches  given 
off  by  the  central  coiled  hypha.  Inside  each  ascus  eight  asco- 
spores  are  produced.  Ultimately  all  the  structures  lying  within 
the  perithecium,  save  the  spores,  undergo  disintegration,  so  that 
the  mature  perithecium  consists  of  a  small  hollow  sphere  within 
which  lie  the  loose  spores.  These  latter  are  ultimately  freed  by 
the  decay  of  the  wall  of  the  perithecium  and  develop  into  new 
individuals.  The  commonest  method  of  reproduction  is  by  the 
formation  of  spores  (gonidia  or  conidia),  which  are  clearly  of 
non-sexual  origin.  These  are  formed  externally  in  the  hyphae 
and  not  inside  sporangia.  A  filament  grows  out,  and  at  its  ter- 
mination a  club-shaped  swelling  is  formed  on  which  a  series  of 
flask-shaped  cells,  called  sterigmata  (vide  Fig.  61,  C6),  are 
perched.  At  the  free  end  of  each  of  these,  an  oval  body,  the 
spore  or  gonidium  is  formed,  and  this  becoming  free,  can  give 
rise  to  a  new  individual. 

(2)  Penicillium  Glaucum. — This  is  perhaps  the  most  com- 
mon of  all  the  fungi  met  with  in  bacteriological  work.  It  is 
the  common  green  cheese  mould,  and  its  spores  are  practically 
omnipresent.  The  mycelium  is  like  that  of  the  aspergillus. 
Perithecium  formation  takes  place,  but  the  commonest  mode  of 
reproduction  is  by  gonidia  (vide  Fig.  61,  D).  A  filament  (called 
a  gonidiophore)  grows  out,  and  at  its  end  breaks  up  into  a  num- 


152          FUNGI:    NON-PATHOGENIC   AND    PATHOGENIC. 

ber  of  finger-like  branches.  On  the  point  of  each  of  these  a 
flask-shaped  sterigma  is  developed.  On  the  end  of  this  a  row 
of  oval  spores  appears.  These  break  off,  and  can  give  rise  to 
new  individuals. 

Yeasts  and  Torulae  :  Saccharomyces,  Torula,  Mycoderma.  - 
These  are  of  the  greatest  importance,  of  course,  in  brewing 
and  baking.  They  only  concern  us  as  being  of  not  uncommon 
occurrence  in  the  air.  They  consist  of  round  or  oval  cells 
usually  many  times  larger  than  bacteria.  They  often  reproduce 
themselves  by  budding  (vide  Fig.  6£,  E,  F),  a  portion  of  the  cell 
protruding,  and  finally  being'  cut  off  to  form  a  new  individual. 
Endogenous  spore  formation  also  occurs  (vide  Fig.  60,  G). 
Many  of  the  torulae,  when  growing  in  colonies,  are  brilliantly 
coloured.  What  their  true  morphological  relationships  are  it  is 
difficult  to  say,  but  they  present  many  analogies  to  the  oidia  of 
such  forms  as  oidium  lactis. 

A  knowledge  of  the  above  type  forms  will  enable  the  student 
to  recognise  the  more  common  fungi  as  such,  when  they  present 
themselves  to  him.  For  further  information  on  this  group  he  is 
referred  to  De  Bary's  book  on  The  Fungi.  Certain  fungi 
closely  related  to  the  above  are  pathogenic  agents.  Some 
aspergilli  have  been  found  to  grow  in  the  animal  tissues  and  to 
produce  death,  and  to  the  fungi  also  belong  the  saprolegniS 
ferax  (tfte  cause  of  a  disease  of  salmon),  the  tinea  tonsurans 
and  the  Achorion  Schoenleinii. 

Blastomycetic  Dermatitis.  —  In  America  and  Germany  within 
recent  years  attention  has  been  called  by  Gilchrist,  Busse,  Stokes, 
Schenck,  Ophiils,  Montgomery,  and  many  others,  to  a  class  of 
diseases  of  the  skin,  resembling  epithelioma  and  tuberculosis, 
which,  in  the  course  of  development,  may  become  generalised 
throughout  the  body  and  result  fatally,  and  post  mortem  show- 
ing more  or  less  extensive  tuberculoid  conditions  of  the  internal 
organs  and  tissues.  In  the  pus  of  abscesses  and  in  sections  of 
diseased  tissue  from  all  cases  reported,  round  bodies  with  doubly 
contoured  walls,  occasionally  budding,  and  closely  resembling 
yeast  cells,  have  been  described,  and  cultures  from  most  of  the 
cases  have  yielded  a  blastomycetic  organism  which  undoubtedly 
is  identical  to  those  bodies  seen  in  the  tissues.  Although 
presenting  some  variations  amongst  themselves  in  culture  media, 
Ricketts  believes  them  to  be  of  very  closely  related  species  of 


BLASTOMYCETIC    DERMATITIS. 


153 


the  genus  oidia,  excepting  those  of  Schenck  and  Hektoen,  which 

have  been  classified  by  Erwin  Smith  as  belonging  to  the  genus 

Sporotricha,     and 

named  by  him  Spo- 

rothrix  Schenckii. 
Morphology.- —  As 

seen    in    the   pus   or 

tissues  (Fig.  62)  these 

bodies    appear    usu- 
ally     as      spherical 

cells,    measuring    in 

diameter     10-15     P> 

possessed   of    a   cell 

membrane  about  .5— 

1.5    //-   in    thickness, 

their     contents     are 

made    up    of    small 

and    large    granules 

of    varying    degrees 

of   refractibility   and 

vacuoles.     No  nuclei 

have  been  demonstrated.     Budding  forms  (Fig.  63),  resembling 

those  of  ordinary  yeast 
cells,  are  often  met 
with,  and  it  would 
appear  as  if  budding 
were  the  usual  method 
of  reproduction;  some 
cells,  however,  contain 
small  round  bodies, 
which  some  have  con- 
sidered endospores 
(Fig.  64),  but  when 
brought  into  cultural 
conditions  these  bod- 
ies have  remained 
unaltered,  and  thus 

FIG.  63.  —  Blastomyces  dermatitidis  (Gilchrist)  :  show  it  IS  extremely  doilbt- 
ingthe  budding  form  of  the  organism  lying  in  the  pus  of  ful  jf  ffrev  have  anv 
an  abscess  cavity  x  1000.  [By  the  kind  permission  of  Dr. 

T.  Caspar  Gilchrist.]  reproductive  f  unction 


FIG.  62.  —  Blastomyces  dermatitidis  (Gilchrist):  sec- 
tion through  an  abscess  cavity  showing  the  organism  with 
doubly-contoured  membrane  X  1000.  [By  the  kind  per- 
mission of  Dr.  T.  Caspar  Gilchrist.J 


154 


FUNGI:    NON-PATHOGENIC   AND   PATHOGENIC. 


whatever.     Involution  forms  are  occasionally  encountered  and 
appear  as  shrunken  empty  capsules  of  irregular  contour.  .   No 

mycelial  development 
has  ever  been  found 
in  the  lesions.  In 
artificial  conditions, 
such  as  afforded  by 
various  cultural  media, 
theorganisms  undergo 
considerable  mo'difica- 
tions.  Three  types  of 
cell  growth  can  be  dis- 
tinguished :  ( i ) round 
or  oval  budding  cells 
much  smaller  than 
those  seen  in  the  tis- 
sues; (2)  segmented, 
branching  mycelial 

FIG.  64.  -Blastomyces   dermatitidis   (Gilchrist)  :    the  threads,     occasionally 

sporulating(?)  form  in  an  abscess  cavity,  showing  eruption  buciclinp;  '       (  3)     aerial 
of  spores  (?)  through  a  rent  in  the  cell  membrane  X  1000.  °  ' 

[By  the  kind  permission  of  Dr.  T.  Caspar  Gilchrist.]  hyphae       bearing      CO- 

nidia. 

Cultural  Characters.  — Ricketts  in  a  comparative  study  divides 
the  organisms  into  three  groups:  (i)  those  growing  upon 
slanted  agar  or  beerwort  agar  with  a  moist,  smooth,  whitish, 
pasty  surface,  and  usually  appearing  upon  the  inoculated  medium 
within  24-48  hours;  (2)  those  having  a  granular,  semi-moist 
surface,  slightly  elevated,  and  incorporated  with  the  medium 
-  after  a  time  the  surface  of  the  growth  becomes  markedly 
plicated  ;  (3)  those  at  first  scarcely  rising  above  the  surface,  deli- 
cate, feathery,  radiate,  semi-moist,  and  of  a  gray-white  colour, 
closely  incorporated  with  the  medium,  and  after  several  days' 
growth 'developing  aerial  hyphae  with  conidia,  in  some  cases, 
resembling  the  growth  of  a  mould,  and  in  the  others  being  dry 
and  as  if  dusted  with  flour.  The  two  latter  groups  require  from 
2-10  days  after  the  medium  has  been  inoculated  before  growth 
develops.  Gilchrist  is  of  the  opinion  that  these  last  two  groups 
of  Ricketts  are  identical,  as  he  has  observed  both  types  of  growth 
occurring  in  the  same  culture  at  different  times.  In  liquid  media 
the  organisms  of  the  first  type  grow  at  the  bottom  of  the  tube 


METHOD    OF   EXAMINATION.  155 

in  a  flocculent  manner,  leaving  the  fluid  clear  ;  the  second  variety 
produces  a  membranous  top  growth,  with  irregular  masses  of 
round  cells  and  mycelial  tufts  at  the  bottom  or  adhering  to  the 
sides  of  the  tube,  the  membrane  so  formed  may  through  increas- 
ing weight  sink  and  be  replaced  by  a  new  formation ;  the  third 
type  forms  no  top  growth,  nor  clouds  the  medium,  but  grows  as 
small,  discrete  woolly  tufts  at  the  bottom.  On  agar  or  gelatin 
.plates  the  colonies  of  the  first  type  are  round,  moist,  glossy, 
white,  opaque,  and  elevated,  and  under  the  low  power  of  the 
microscope  are  found  to  be  coarsely  granular,  and  individual  cells 
can  be  seen  at  their  peripheries.  The  second  type  shows  round- 
ish colonies  having  the  characters  described  upon  agar  slants ; 
microscopically  the  colonies  are  made  up  of  branching  segmented 
mycelium  radiating  outwards  from  the  centre  of  the  colony.  In 
the  third  type  the  colonies  have  the  general  characters  of  the 
slant  agar  growth,  arid  under  the  microscope  appear  to  be  made 
up  of  long  radiating,  branching  segmented  mycelium,  which  here 
and  there  shows  occasional  budding. 

Fermentation  of  glucose  and  maltose  with  the  production  of 
alcohol,  acetic  acid,  and  carbon  dioxide  takes  place  with  some 
varieties,  whilst  in  others  no  fermentation  occurs  at  all.  Cane 
sugar  and  lactose  are  not  fermented  by  any  variety. 

Litmus  milk  is  rendered  usually  neutral  or  alkaline  by  the 
growth,  whilst  acidity  and  coagulation  of  the  casein  is  very  rare. 

Gelatin  in  some  instances  is  liquefied  slowly. 

Pathogenic  Properties.  —  Towards  the  lower  animals  patho- 
genic properties  vary  very  much  with  the  culture,  recently 
isolated  cultures  as  a  rule  proving  more  virulent  than  older 
ones.  Mice,  guinea-pigs,  and  dogs  are  most  susceptible,  suc- 
cumbing often  to  subcutaneous  and  intraperitoneal  inoculations, 
whilst  the  white  rat,  rabbit,  sheep,  and  horse  are  more  refrac- 
tory; in  all,  the  lesions  may  be  localized  in  the  form  of  abscesses, 
or  general  infection  may  ensue  where  subcutaneous  inoculation 
is  practised. 

Methods  of  Examination  and  Isolation.  —  Pus  from  subcu- 
taneous abscesses,  or  scrapings  made  with  a  curette  from  the 
inflamed  indurated  skin  growth  are  to  be  placed  on  a  slide  and 
thoroughly  macerated  in  a  thirty  per  cent  solution  of  sodium 
hydrate,  which  dissolves  the  pus  or  tissue  cells  and  permits  of 
the  more  ready  identification  of  the  blastomycetes,  which  are 


156         FUNGI:    NON-PATHOGENIC   AND   PATHOGENIC. 

recognized  by  their  large  size,  doubly  contoured  membrane, 
and  refractile  granular  contents,  and  perhaps  by  evidences  of 
budding  at  some  portion  of  the  cell  wall.  Staining  of  the  pus 
or  scrapings  is  not  so  satisfactory  as  an  examination  of  the  fresh 
material,  but  this  can  be  carried  out  by  making  film^  and  stain- 
ing in  an  old  solution  of  Loffler's  methylene-blue  or  by  Gram's 
method,  as  described  in  Chapter  III. 

Isolation  in  pure  culture  is  most  readily  effected  by  the  use 
of  saccharine  media,  such  as  glucose  or  maltose  broth  (or  agar), 
beerwort  or  beerwort  agar  or  gelatin  (see  Chapter  II.),  yet  in 
spite  of  all  efforts  some  cases  yield  negative  results.  Inoculation 
of  the  media  is  made  from  pus,  scrapings  of  the  growth,  or  even 
with  very  small  pieces  of  excised  tissue  from  the  growing  edge 
of  the  lesion,  and  incubation  at  37°  C.  is  to  be  prolonged  for  two 
weeks  before  any  culture  is  declared  negative.  The  appear- 
ance of  round  or  oval,  budding  and  non-budding  cells,  alone 
or  mixed  with  branching  or  budding  mycelial  threads,  together 
with  general  characters  described  above,  may  be  regarded  as 
of  a  positive  nature.  Animal  inoculations  should  likewise  be 
carried  out. 


I/ 


CHAPTER   VI. 

RELATIONS   OF   BACTERIA  TO    DISEASE— THE   PRODUCTION 
OF   TOXINS    BY    BACTERIA. 

Introductory.  —  It  has  already  been  stated  that  a  strict  divi- 
sion of  micro-organisms  into  saprophytes  and  true  parasites  can- 
not be  made.  No  doubt  there  are  organisms,  such  as  the 
bacillus  of  leprosy  and  the  spirillum  of  relapsing  fever,  which 
as  yet  have  not  been  cultivated  outside  the  animal  body,  and 
others,  such  as  the  gonococcus,  which  are  in  natural  conditions 
always  parasites  associated  with  disease.  But  these  latter  can 
lead  a  saprophytic  existence  in  specially  prepared  conditions, 
and  there  are  many  of  the  disease-producing  organisms,  such  as 
the  organisms  of  typhoid  and  cholera,  which  can  flourish  readily 
outside  the  body,  even  in  ordinary  conditions.  The  conditions 
of  growth  are,  however,  of  very  great  importance  in  the  study 
of  the  modes  of  infection  in  the  various  diseases,  though  they 
do  not  form  the  basis  of  a  scientific  division. 

A  similar  statement  applies  to  the  terms  pathogenic  and 
sapropJiytic,  and  even  to  the  terms  pathogenic  and  non-pathogenic. 
By  the  term  pathogenic  is  meant  the  power  which  an  organism 
has  of  producing  morbid  changes  or  effects  in  the  animal  body, 
either  under  natural  conditions  or  in  conditions  artificially 
arranged,  as  in  direct  experiment.  Now  we  know  of  no  organ- 
isms which  will  in  all  circumstances  produce  disease  in  all 
animals,  and,  on  the  other  hand,  many  bacteria  described  as 
harmless  saprophytes  will  produce  pathological  changes  if  intro- 
duced in  sufficient  quantity.  When,  therefore,  we  speak  of  a 
pathogenic  organism,  the  term  is  merely  a  relative  one,  and 
indicates  that  in  certain  circumstances  the  organism  will  pro- 
duce disease,  though  in  the  science  of  human  pathology  it  is 
often  used  for  convenience  as  implying  that  the  organism  pro- 
duces disease  in  man  in  natural  conditions. 

157 


158  RELATIONS   OF    BACTERIA   TO   DISEASE. 

Modifying  Conditions. — In  studying  the  pathogenic  effects  in 
any  instance,  both  'the  micro-organisms  and  the  animal  affected 
must  be  considered,  and  not  only  the  species  of  each,  but  also 
its  exact  condition  at  the  time  of  infection.  In  other  words, 
the  resulting  disease  is  the  product  of  the  sum  total  of  the 
characters  of  the  infecting  agent,  on  the  one  hand,  and  of  the 
subject  of  infection,  on  the  other.  We  may,  therefore,  state 
some  of  the  chief  circumstances  which  modify  each  of  these 
two  factors  involved  and,  consequently,  the  diseased  condition 
produced. 

i.  The  Infecting  Agent.  —  In  the  case  of  a  particular  species 
of  bacterium  its  effect  will  depend  chiefly  upon  (a)  its  virulence, 
and  (U)  the  number  introduced  into  the  body.  To  these  may 
be  added  (c)  the  path  of  infection. 

The  virulence,  i.e.  the  power  of  multiplying  in  the  body,  and 
producing  disease,  varies  greatly  in  different  conditions,  and  the 
methods  by  which  it  can  be  diminished  or  increased  will  be 
afterwards  described  (vide  Chapter  XX.).  One  important 
point  is  that  when  a  bacterium  has  been  enabled  to  invade 
and  multiply  in  the  tissues  of  an  animal,  its  virulence  for  that 
species  is  often  increased.  This  is  well  seen  in  the  case  of 
certain  bacteria  which  are  normally  present  on  the  skin  or 
mucous  surfaces.  Thus  it  has  been  repeatedly  proved  that  the 
bacillus  coli  cultivated  from  a  septic  peritonitis  is  much  more 
virulent  than  that  taken  from  the  bowel  of  the  same  animal. 
The  virulence  may  be  still  more  increased  by  inoculating  from 
one  animal  to  another  in  series  —  the  method  of  passage.  Widely 
different  effects  are,  of  course,  produced  on  the  virulence  being 
altered.  For  example,  a  streptococcus  which  produces  merely 
a  local  inflammation  or  suppuration  may  produce  a  rapidly 
fatal  septicaemia  when  its  virulence  is  raised.  Virulence  also 
has  a  relation  to  the  animal  employed,  as  occasionally  on  being 
increased  for  one  species  of  animal  it  is  diminished  for  another. 
For  example,  streptococci,  on  being  inoculated  in  series  through 
a  number  of  mice,  acquire  increased  virulence  for  these  animals, 
but  become  less  virulent  for  rabbits.  (Knorr.) 

The  number  of  the  organisms  introduced,  i.e.  the  dose  of  the 
infecting  agent,  is  another  point  of  importance.  The  healthy 
tissues  can  usually  resist  a  certain  number  of  pathogenic  organ- 
isms of  given  virulence,  and  it  is  only  in  a  few,  instances  that 


CONDITIONS   MODIFYING   PATHOGENICITY.  159 

one  or  two  organisms  introduced  will  produce  a  fatal  disease, 
e.g.  the  case  of  anthrax  in  white  mice.  The  healthy  peritoneum 
of  a  rabbit  can  resist  and  destroy  a  considerable  number  of 
pyogenic  micrococci  without  any  serious  result,  but  if  a  larger 
dose  be  introduced,  a  fatal  peritonitis  may  follow.  Again,  a 
certain  quantity  of  a  particular  organism  injected  subcutaneously 
may  produce  only  a  local  inflammatory  change,  but  in  the  case 
of  a  larger  dose  the  organisms  may  gain  entrance  to  the  blood 
stream  and  produce  septicaemia.  There  is,  therefore,  for  a  par- 
ticular animal,  a  minimum  lethal  dose  which  can  be  determined 
by  experiment  only ;  a  dose,  moreover,  which  is  modified  by 
various  circumstances  difficult  to  control. 

The  path  of  infection  may  alter  the  result,  serious  effects  often 
following  a  direct  entrance  into  the  blood  stream.  Staphylo- 
cocci  injected  subcutaneously  in  a  rabbit  may  produce  only  a 
local  abscess,  whilst  on  intravenous  injection  multiple  abscesses 
in  certain  organs  may  result  and  death  may  follow.  Local 
inflammatory  reaction  with  subsequent  destruction  of  the  organ- 
isms may  be  restricted  to  the  site  of  infection  or  may  occur  also 
in  the  lymphatic  glands  in  relation.  The  latter  therefore  act  as 
a  second  barrier  of  defence,  or  as  a  filtering  mechanism  which 
aids  in  protecting  against  blood  infection.  This  is  well  illus- 
trated in  the  case  of  "poisoned  wounds."  In  some  other  cases, 
however,  the  organisms  are  very  rapidly  destroyed  in  the  blood 
stream,  and  Klemperer  has  found  that,  in  the  dog,  subcutaneous 
injection  of  the  pneumococcus  produces  death  more  readily  than 
intravenous  injection. 

2.  The  Subject  of  Infection.  —  Amongst  healthy  individuals 
susceptibility  and,  in  inverse  ratio,  resistance  to  a  particular 
microbe  may  vary  according  to  (a)  species,  (b)  race  and  indi- 
vidual peculiarities,  (c)  age.  Different  species  of  the  lower 
animals  show  the  widest  variation  in  this  respect,  some  being 
extremely  susceptible,  others  highly  resistant.  Then  there  are 
diseases,  such  as  leprosy,  gonorrhoea,  etc.,  which  appear  to  be 
peculiar  to  the  human  subject  and  have  not  yet  been  transmitted 
to  animals.  And  further,  there  are  others,  such  as  cholera  and 
typhoid,  which  do  not  naturally  affect  animals,  and  the  typical 
lesions  of  which  cannot  be  experimentally  reproduced  in  them, 
or  appear  only  imperfectly,  although  pathogenic  effects  follow 
inoculation  with  the  organisms.  In  the  case  of  the  human  sub- 


160  RELATIONS   OF   BACTERIA  TO    DISEASE. 

ject,  differences  in  susceptibility  to  a  certain  disease  are  found 
amongst  different  races  and  also  amongst  individuals  of  the 
same  race,  as  is  well  seen  in  the  case  of  tubercle  and  other 
diseases.  Age  also  plays  an  important  part,  young  subjects 
being  more  liable  to  certain  diseases,  e.g.  to  diphtheria.  Further, 
at  different  periods  of  life  certain  parts  of  the  body  are  more 
susceptible,  for  example,  in  early  life,  the  bones  and  joints  to 
tubercular  and  acute  suppurative  affections. 

In  increasing  the  susceptibility  of  a  given  individual,  condi- 
tions of  local  or  general  diminished  vitality  play  the  most  important 
part.  It  has  been  experimentally  proved  that  conditions  such  as 
exposure  to  cold,  fatigue,  starvation,  etc.,  all  diminish  the  natural 
resistance  to  bacterial  infection.  Rats  naturally  immune  can  be 
rendered  susceptible  to  glanders  by  being  fed  with  phloridzin, 
which  produces  a  sort  of  diabetes,  a  large  amount  of  sugar  being 
excreted  in  the  urine  (Leo).  Guinea-pigs  may  resist  subcuta- 
neous injection  of  a  certain  dose  of  the  typhoid  bacillus,  but  if 
at  the  same  time  a  sterilised  culture  of  the  bacillus  coli  be 
injected  into  the  peritoneum,  they  quickly  die  of  a  general 
infection.  Also  a  local  susceptibility  may  be  produced  by  injur- 
ing or  diminishing  the  vitality  of  a  part.  If,  for  example,  pre- 
vious to  an  intravenous  injection  of  staphylococci,  the  aortic 
cusps  of  a  rabbit  be  injured,  the  organisms  may  settle  there  and 
set  up  an  ulcerative  endocarditis ;  or  if  a  bone  be  injured,  they 
may  produce  suppuration  at  the  part,  whereas  in  ordinary  cir- 
cumstances these  lesions  would  not  take  place.  The  action  of 
one  species  of  bacterium  is  also  often  aided  by  the  simultaneous 
presence  of  other  species.  In  this  case  the  latter  may  act  simply 
as  additional  irritants  which  lessen  the  vitality  of  the  tissues,  but 
in  some  cases  their  presence  also  appears  to  favour  the  develop- 
ment of  a  higher  degree  of  virulence  of  the  former. 

These  facts,  established  by  experiment  (and  many  others 
might  be  given),  illustrate  the  important  part  which  local  or 
general  conditions  of  diminished  vitality  may  play  in  the  pro- 
duction of  disease  in  the  human  subject.  This  has  long  been 
known  by  clinical  observation.  In  normal  conditions  the  blood 
and  tissues  of  the  body,  with  the  exception  of  the  skin  and  cer- 
tain of  the  mucous  surfaces,  are  bacterium-free,  and  if  a  few 
organisms  gain  entrance,  they  are  destroyed.  But  if  the  vitality 
becomes  lowered  their  entrance  becomes  easier  and  the  pos- 


MODES   OF   BACTERIAL   ACTION.  161 

sibility  of  their  multiplying  and  producing  disease  greatly 
increased.  In  this  way  the  favouring  part  played  by  fatigue, 
cold,  etc.,  in  the  production  of  diseases  of  which  the  direct  cause 
is  a  bacterium,  may  be  understood.  It  is  important  to  keep  in 
view  in  this  connection  that  many  of  the  inflammation-producing 
and  pyogenic  organisms  are  normally  present  on  the  skin  and 
various  mucous  surfaces.  The  action  of  a  certain  organism 
may  devitalise  the  tissues  to  such  an  extent  as  to  pave  the  way 
for  the  entrance  of  other  bacteria  ;  we  may  mention  the  liability 
of  the  occurrence  of  pneumonia,  erysipelas,  and  various  suppura- 
tive  conditions  in  the  course  of  or  following  infective  fevers. 
In  some  cases  the  specific  organism  may  produce  lesions  through 
which  the  other  organisms  gain  entrance,  e.g.  in  typhoid,  diph- 
theria, etc.  It  is  not  uncommon  to  find  in  the  bodies  of  those 
who  have  died  from  chronic  wasting  disease,  collections  of 
micrococci  or  bacilli  in  the  capillaries  of  various  organs,  which 
have  entered  in  the  later  hours  of  life;  that  is  to  say,  the 
bacterium-free  condition  of  the  blood  has  been  lost  in  the  period 
of  prostration  preceding  death. 

The  methods  by  which  the  natural  resistance  may  be  specifi- 
cally increased  belong  to  the  subject  of  immunity,  and  are 
described  in  the  chapter  on  that  subject. 

Modes  of  Bacterial  Action.  —  In  the  production  of  disease 
by  micro-organisms  there  are  two  main  factors  involved,  namely, 
(a)  the  multiplication  of  the  living  organisms  after  they  have 
entered  the  body,  and  (b),  the  production  by  them  of  poisons 
which  may  act  both  upon  the  tissues  around  and  upon  the  body 
generally.  The  former  corresponds  to  infection,  the  latter  is  of 
the  nature  of  intoxication  or  poisoning.  In  different  diseases 
one  of  these  is  usually  the  more  prominent  feature,  but  both  are 
always  more  or  less  concerned. 

I .  Infection  and  Distribution  of  the  Bacteria  in  the  Body.  — 
After  pathogenic  bacteria  have  invaded  the  tissues,  or  in  other 
words  after  infection  by  bacteria  has  taken  place,  their  further 
behaviour  varies  greatly  in  different  cases.  In  certain  cases 
they  may  reach  and  multiply  in  the  blood  stream,  producing  a 
fatal  septicaemia.  In  the  lower  animals  this  multiplication  of  the 
organisms  in  the  blood  throughout  the  body  may  be  very  exten- 
sive (for  example,  the  septicaemia  produced  by  the  pneumococcus- 
in  rabbits) ;  but  in  septicaemia  in  man,  it  very  seldom,  if  ever, 


162  RELATIONS    OF    BACTERIA   TO    DISEASE. 

occurs  to  so  great  a  degree,  the  organisms  rarely  remain  in  large 
numbers  in  the  circulating  blood,  and  their  detection  in  it  during 
life  by  microscopic  examination  is  rare,  and  even  culture  methods 
may  give  negative  results  unless  a  large  amount  of  blood  is  used, 
yet  it  is  interesting  to  note  that  the  researches  of  Schottmiiller, 
Auerbach  and  Unger,  Cole  and  others,  show  that  in  various 
septicaemias,  pneumonia,  and  typhoid  fever  the  blood  may  con- 
tain at  certain  periods  of  the  diseases  the  specific  micro-organ- 
isms in  a  relatively  large  percentage  of  cases. 

However,  in  fatal  cases  where  blood  cultures  are  negative 
during  life,  the  organisms  may  be  found  post  mortem  lying  in 
large  numbers  within  the  capillaries  of  various  organs,  e.g.  in 
cases  of  septicaemia  produced  by  streptococci.  (Relapsing  fever 
forms  an  exception,  as  in  it  numerous  organisms  may  be  seen  in 
a  drop  of  blood.)  In  the  human  subject  more  frequently  one 
of  two  things  happens.  In  the  first  place,  the  organisms  may 
remain  local,  producing  little  reaction  around  them,  as  in  teta- 
nus, or  a  well-marked  lesion,  as  in  diphtheria,  pneumonia,  etc. 
Or,  in  the  second  place,  they  may  pass  by  the  lymph  or  blood 
stream,  to  other  parts  or  organs  in  which  they  settle,  multiply, 
and  produce  lesions,  as  in  tubercle. 

2.  Production  of  Chemical  Poisons.  —  In  all  these  cases  the 
growth  of  the  organisms  is  accompanied  by  the  formation  of 
chemical  products,  which  act  generally  or  locally  in  varying 
degree  as  toxic  substances.  The  toxic  substances  become  dif- 
fused throughout  the  system,  and  their  effects  are  manifested 
chiefly  by  symptoms  such  as  the  occurrence  of  fever,  disturb- 
ances of  the  circulatory,  respiratory,  and  nervous  systems,  etc. 
In  some  cases  corresponding  changes  in  the  tissues  are  found, 
for  example,  the  changes  in  the  nervous  system  in  diphtheria,  to 
be  afterwards  described.  The  general  toxic  effects  may  be  so 
slight  as  to  be  of  no  importance,  as  in  the  case  of  a  local  suppu- 
ration, or  they  may  be  very  intense,  as  in  tetanus,  or  again,  less 
severe  but  producing  cachexia  by  their  long  continuance,  as  in 
tuberculosis. 

The  occurrence  of  local  tissue  changes  or  lesions  produced  in 
the  neighbourhood  of  the  bacteria,  as  already  mentioned,  is  one 
of  the  most  striking  results  of  bacterial  action,  but  these  also 
must  be  traced  to  chemical  substances  formed  in  or  around  the 
bacteria,  and  either  directly  or  through  the  medium  of  ferments. 


PRODUCTION   OF   CHEMICAL   POISONS.  163 

In  this  case  it  is  more  difficult  to  demonstrate  the  mode  of 
action,  for  in  the  tissues  the  chemical  products  are  formed  by 
the  bacteria  slowly,  continuously,  and  in  a  certain  degree  of 
concentration,  and  these  conditions  cannot  be  exactly  reproduced 
by  experiment.  It  is  also  to  be  noted  that  more  than  one  poison 
may  be  produced  by  a  given  bacterium.  Thus  in  diphtheria, 
for  example,  the  products  which  produce  the  local  inflammatory 
reaction  and  necrosis  are  in  all  probability  not  the  same  as  those 
which  affect  the  nerve  cells  and  fibres.  Further,  it  is  very 
doubtful  whether  all  the  chemical  substances  formed  by  a  cer- 
tain bacillus  growing  in  the  tissues  are  also  formed  by  it  in 
cultures  outside  the  body.  The  separated  toxin  of  diphtheria, 
like  various  vegetable  and  animal  toxins  (vide  infra),  however, 
possesses  a  local  toxic  action  of  very  intense  character,  often 
producing  extensive  necrotic  change. 

The  injection  of  large  quantities  of  many  different  pathogenic 
organisms  in  the  dead  conditions  results  in  the  production  of  a 
local  inflammatory  change  which  may  be  followed  by  suppura- 
tion, this  effect  being  possibly  brought  about  by  certain  sub- 
stances in  the  bacterial  protoplasm  common  to  various  species, 
or  at  least  possessing  a  common  physiological  action  (Buchner 
and  others).  When  dead  tubercle  bacilli,  however,  are  intro- 
duced into  the  blood  stream,  nodules  do  result  in  certain  parts 
which  have  a  resemblance  to  ordinary  tubercles.  In  this  case 
the  bodies  of  the  bacilli  evidently  contain  a  highly  resistant  and 
slowly  acting  substance  which  gradually  diffuses  around  and 
produces  effects  (vide  "  Tuberculosis  "). 

The  action  of  bacteria  as  mechanical  irritants  plays  a  very 
small  part  in  the  processes  of  disease ;  and  the  differences  in 
their  effects,  though  regulated  by  the  position  and  rate  of 
growth  of  the  organisms,  can  be  accounted  for  only  by  the 
formation  of  definite  chemical  substances  which  act  on  the 
tissues. 

Summary.  —  We  may  say  then  that  the  action  of  bacteria  as 
disease-producers,  as  in  fact  their  power  to  exist  and  multiply  in 
the  living  body,  depends  upon  the  chemical  preducts  formed 
directly  or  indirectly  by  them.  This  action  is  shown  by  tissue 
changes  produced  in  the  vicinity  of  the  bacteria  or  throughout 
the  system,  and  by  toxic  symptoms  of  great  variety  of  degree  and 
character. 


164  RELATIONS    OF   BACTERIA   TO    DISEASE. 

We  shall  first  consider  the  effects  of  bacteria  on  the  body 
generally,  and  afterwards  the  nature  of  the  chemical  products. 

EFFECTS  OF  BACTERIAL  ACTION. 

These  may  be  for  convenience  arranged  in  a  tabular  form 
as  follows :  — 

A.  Tissue  Changes. 

(1)  Local  changes,  i.e.  changes  produced  in  the  neigh- 

bourhood of  the  bacteria. 
Position  (a]  At  primary  lesion. 
(&)  At  secondary  foci. 

Character  (a)  Tissue  reactions  1  Acute  or 

(b)  Degeneration  and  necrosis  j  Chronic. 

(2)  Produced  at  a  distance  from  the  bacteria,  directly 

or  indirectly,  by  the  absorption  of  toxins. 

(a)  In  special  tissues  — 

(a)  as  the  result  of  damage,  e.g.  nerve  cells 
and  fibres,  secreting  cells,  vessel  walls, 
or 

(/3)  changes  of  a  reactive  nature  in  the  blood- 
forming  organs. 

(b)  General  anatomical  changes,  the  effects  of  mal- 

nutrition or  of  increased  waste. 

B.  Changes  in  Metabolism. 

The  occurrence  of  fever,  of  errors  of  assimilation  and 
elimination,  etc. 

A.  Tissue  Changes  produced  by  Bacteria.  —  The  effects  of 
bacterial  action  are  so  various  as  so  include  almost  all  known 
pathological  changes.  However  varied  in  character,  they  may 
be  classified  under  two  main  headings  —  (a)  those  of  a  degen- 
erative or  necrotic  nature,  the  direct  result  of  damage,  and  (b} 
those  of  reactive  nature,  defensive  or  reparative.  The  former  are 
the  expression  of  the  necessary  vulnerability  of  the  tissues,  the 
latter  of  protective  powers  evolved  for  the  benefit  of  the  organism. 
In  the  means  of  defence,  both  leucocytes  and  the  fixed  cells  of 
the  tissues  are  concerned.  Both  show  phagocytic  properties,  i.e. 
have  the  power  of  taking  up  bacteria  into  their  protoplasm.  The 


TISSUE   CHANGES   PRODUCED   BY   BACTERIA.  165 

cells  are  guided  towards  the  focus  of  infection  by  chemiotaxis, 
and  thus  we  find  that  different  bacteria  attract  different  cells. 
The  most  rapid  and  abundant  supply  of  phagocytes  is  seen  in 
the  case  of  suppurative  conditions,  where  the  neutrophile  leuco- 
cytes of  the  blood  are  chiefly  concerned.  When  the  local  lesion 
is  of  some  extent  there  is  usually  an  increase  of  these  cells  in 
the  blood  —  a  neutrophile  leucocytosis.  And  further,  recent 
observations  have  shown  that  associated  with  this  there  is  in  the 
bone-marrow  an  increased  number  of  the  mother-cells  of  these 
leucocytes  —  the  neutrophile  myelocytes.  The  passage  of  the 
neutrophile  leucocytes  from  the  marrow  into  the  blood,  with  the 
resulting  leucocytosis,  is  also  apparently  due  to  the  absorbed 
bacterial  toxins  acting  chemiotactically  on  the  marrow.  These 
facts  abundantly  show  that  the  means  of  defence  is  not  a  mere 
local  mechanism,  but  that  increased  proliferative  activity  in 
distant  tissues  is  called  into  play.  In  addition  to  direct  phagocy- 
tosis by  these  leucocytes,  there  is  now  abundant  evidence  that  an 
important  function  is  the  production  in  the  body  of  bactericidal 
and  other  antagonistic  substances.  In  other  cases  the  cells 
chiefly  involved  are  the  mononuclear  hyaline  leucocytes,  and 
with  them  the  endothelial  cells,  e.g.  of  serous  membranes,  often 
play  an  important  part  in  the  defence ;  this  is  well  seen  in 
typhoid  fever,  where  the  specific  bacillus  appears  to  have  little 
or  no  action  on  the  neutrophile  leucocytes.  In  other  cases, 
again,  the  reaction  is  chiefly  on  the  part  of  the  connective 
cells,  though  their  proliferation  is  always  associated  with  some 
variety  of  leucocytic  infiltration  and  usually  also  with  the  forma- 
tion of  new  blood-vessels.  Such  a  connective-tissue  reaction 
occurs  especially  in  slow  infections  or  in  the  later  stages  of  an 
acute  infection.  The  tissue  changes  resulting  from  cellular 
activity  in  the  presence  of  bacterial  invasion  are  naturally  very 
varied  —  of  this  examples  will  be  found  in  subsequent  chapters ; 
but  they  may  be  said  to  be  manifestations  of  the  two  funda- 
mental processes  of  (a)  increased  functional  activity — movement, 
phagocytosis,  secretion,  etc.  —  and  (b}  increased  formative  activity 
—  cell  growth  and  division.  The  exudation  from  the  blood- 
vessels has  been  variously  interpreted.  There  is  no  doubt  that 
the  exudate  has  bactericidal  properties  and  also  acts  as  a  diluting 
agent,  but  it  must  still  be  held  as  uncertain  whether  the  process 
of  exudation  ought  to  be  regarded  as  primarily  defensive  or  as 


166  RELATIONS   OF   BACTERIA   TO   DISEASE. 

the  direct  result  of  damage  to  the  endothelium  of  the  vessels. 
It  may  also  be  pointed  out  that  the  various  changes  referred  to 
are  none  of  them  peculiar  to  bacterial  invasion ;  they  are  exam- 
ples of  the  general  laws-  of  tissue  change  under  abnormal  con- 
ditions, and  they  can  all  be  reproduced  by  chemical  substances 
in  solution  or  in  a  particulate  state.  What  constitutes  their 
special  feature  is  their  progressive  or  spreading  nature,  due  to 
the  bacterial  multiplication. 

(i)  Local  Lesions.  —  In  some  diseases  the  lesion  has  a  special 
site;  for  example,  the  lesion  of  typhoid  fever  and,  to  a  less 
extent,  that  of  diphtheria.  In  other  cases  it  depends  entirely 
upon  the  point  of  entrance,  e.g.  malignant  pustule  and  the  con- 
ditions known  as  wound  infections.  In  others,  again,  there  is 
a  special  tendency  for  certain  parts  to  be  affected,  as  the  upper 
parts  of  the  lungs  in  tubercle.  In  some  cases  the  site  has  a 
mechanical  explanation. 

When  organisms  gain  an  entrance  to  the  blood  from  a  pri- 
mary lesion,  directly  or  by  the  lymphatic  system,  they  may 
become  destroyed,  or  they  may  settle  in  certain  organs  and 
produce  their  characteristic  effects.  The  organs  specially 
liable  to  be  affected  in  this  way  vary  in  different  diseases. 
Pyogenic  cocci  show  a  special  tendency  to  settle  in  the  capil- 
laries of  the  kidneys  and  produce  miliary  abscesses,  whilst  these 
lesions  rarely  occur  in  the  spleen.  On  the  other  hand,  the 
nodules  in  disseminated  tubercle  or  glanders  are  much  more 
numerous  in  the  spleen  than  in  the  kidneys,  which  in  the  latter 
disease  are  usually  free  from  them.  The  important  point  is  that 
the  position  of  the  disseminated  lesions  is  not  to  be  explained 
by  a  mechanical  process,  such  as  embolism,  but  depends  upon 
a  special  relation  between  the  organisms  and  the  tissues,  which 
may  be  spoken  of  either  as  a  selective  power  on  the  part  of  the 
organisms  or  a  special  susceptibility  of  tissues,  possibly  in  part 
due  to  their  affording  to  the  organisms  more  suitable  conditions 
of  nutriment.  Even  in  the  case  of  the  lesions  produced  by  dead 
tubercle  bacilli,  a  certain  selective  character  is  observed. 

Acute  Local  Lesions.  —  The  local  inflammatory  reaction  pre- 
sents different  characters  in  different  conditions.  It  may  be 
accompanied  by  abundant  fibrinous  exudation,  or  by  great 
catarrh  (in  the  case  of  an  epithelial  surface),  or  by  haemorrhage, 
or  by  oedema;  it  may  be  localised  or  spreading  in  character;  it 


LOCAL   LESIONS.  167 

may  be  followed  by  suppuration,  or  may  lead  to  necrosis.  A 
few  examples  may  be  given.  A  great  many  different  organ- 
isms cause  an  abundant  fibrinous  exudation.  This,  along  with 
necrosis  of  epithelium,  is  the  action  of  the  diphtheria  bacillus  on 
a  mucous  membrane,  and  also  of  streptococci  in  certain  condi- 
tions ;  it  is  produced  in  the  alveoli  of  the  lung  in  croupous  pneu- 
monia by  the  pneumococcus  and  probably  by  other  organisms, 
whilst  fibrinous  inflammation  in  serous  cavities  is  produced  by  a 
great  many  different  bacteria.  The  last  statement  also  applies 
to  numerous  suppurative  and  catarrhal  conditions.  The  in- 
flammatory change  in  a  Peyer's  patch  in  typhoid  fever,  though 
fibrinous  exudation  is  less  marked,  is  followed  by  necrosis,  while 
in  the  malignant  pustule  of  man  necrotic  change  attended  by 
considerable  haemorrhage  is  one  of  the  chief  features.  The 
great  variety  in  local  reaction  is  well  illustrated  in  the  case  of 
skin  lesions  produced  by  bacteria.  The  necrotic  or  degenerative 
changes  affecting  especially  the  more  highly  developed  elements 
are  chiefly  produced  by  the  direct  action  of  the  bacterial  poisons, 
though  aided  by  the  disturbances  of  nutrition  involved  in  the 
vascular  phenomena. 

In  many  of  the  acute  inflammatory  conditions,  if  not  attended 
by  a  fatal  result,  the  disease  comes  to  a  natural  termination  after 
a  certain  time,  e.g.  in  pneumonia,  erysipelas,  etc.  This  fact, 
the  explanation  of  which  is  not  yet  fully  understood,  has  an 
important  relation  to  the  subject  of  immunity,  and  will  be 
discussed  later.  It  may  also  be  pointed  out  that  a  well-niarked 
inflammatory  reaction  is  often  found  in  animals  which  occupy 
a  medium  position  in  the  scale  of  susceptibility,  and  that  an 
organism  which  causes  a  general  infection  in  a  certain  animal 
may  produce  only  a  local  inflammation  when  its  virulence  is 
lessened. 

Chronic  Local  Lesions.  —  In  a  considerable  number  of  diseases 
produced  by  bacteria  the  local  tissue  reaction  is  a  more  chronic 
process  than  those  described.  In  other  words,  the  specific 
irritant  is  less  intense,  so  that  there  is  less  vascular  disturbance 
and  a  greater  preponderance  of  the  proliferative  processes, 
leading  to  new  formation  of  connective  tissue  or  a  modified 
connective  tissue.  This  formation  may  occur  in  foci  here  and 
there,  so  that  nodules  of  greater  or  less  consistence  result,  or  it 
may  be  more  diffuse.  Such  changes  especially  occur  in  the 


168  RELATIONS   OF   BACTERIA   TO   DISEASE. 

diseases  often  known  as  the  infective  granulomata,  of  which 
tubercle,  leprosy,  glanders,  actinomycosis,  syphilis,  etc.,  are 
examples.  A  hard  and  fast  line,  however,  cannot  be  drawn 
between  such  conditions  and  those  described  above  as  acute. 
In  glanders,  for  example,  especially  in  man,  the  lesion  produced 
by  the  glanders  bacillus  often  approaches  very  nearly  to  an 
acute  suppurative  change,  and  sometimes  actually  is  of  this 
nature.  Whilst  in  these  diseases  the  fundamental  change  is 
the  same,  viz.,  a  reaction  to  an  irritant  of  minor  intensity,  the 
exact  structural  characters  and  arrangement  vary  in  different 
diseases.  In  some  cases  the  disease  may  be  identified  by  the 
histological  changes  alone,  but  on  the  other  hand  this  is  often 
impossible.  These  changes  often  include  the  occurrence  of 
degenerations  or  of  actual  necrosis  in  the  newly  formed  tissue. 
In  the  granulomata,  infection  of  other  parts  from  the  primary 
lesion  takes  place  chiefly  by  the  blood-vessels  and  lymphatics, 
though  sometimes  along  natural  tubes  such  as  the  bronchi, 
intestine,  etc.  The  organs  specially  liable  to  be  the  site  of 
secondary  lesions  vary  in  different  diseases,  as  already  ex- 
plained. 

(2)  General  Lesions  produced  by  Toxins.  —  In  the  various 
infective  conditions  produced  by  bacteria,  changes  commonly 
occur  in  certain  organs  unassociated  with  the  presence  of  the 
bacteria ;  these  are  produced  by  the  action  of  bacterial  products 
circulating  in  the  blood.  Many  such  lesions  can  be  produced 
experimentally.  The  secreting  cells  of  various  organs,  especially 
the  kidney  and  liver,  are  specially  liable  to  change  of  this  kind. 
Cloudy  swelling,  which  may  be  followed  by  fatty  change  or  by 
actual  necrosis  with  granular  disintegration,  is  common.  Hya- 
line change  in  the  walls  of  arterioles  may  occur,  and  in  certain 
chronic  conditions  waxy  change  is  brought  about  in  a  similar 
manner.  The  latter  has  been  produced  in  animals  by  the 
repeated  injection  of  the  staphylococcus  aureus.  Capillary 
haemorrhages  are  not  uncommon,  and  are  in  many  cases  due  to 
an  increased  permeability  of  the  vessel  walls,  aided  by  changes 
in  the  blood  plasma,  as  evidenced  sometimes  by  diminished 
coagulability.  Similar  haemorrhages  may  follow  the  injection  of 
some  bacterial  toxins,  e.g.  of  diphtheria,  and  also  of  vegetable 
poisons,  e.g.  ricin  and  abrin.  Skin  eruptions  occurring  in  the 
exanthemata  are  probably  produced  in  the  same  way,  though 


DISTURBANCES   OF   METABOLISM.  169 

in  many  of  these  diseases  the  causal  organism  has  not  yet  been 
isolated.  We.  have,  however,  the  important  fact  that  corre- 
sponding skin  eruptions  may  be  produced  by  poisoning  with 
certain  drugs.  In  the  nervous  system  degenerative  changes 
have  been  found  in  diphtheria,  both  in  the  spinal  cord  and  in 
the  peripheral  nerves,  and  have  been  reproduced  experimentally 
by  the  products  of  the  diphtheria  bacilli.  There  is  also  experi- 
mental evidence  that  the  bacillus  coli  communis  and  the  strepto- 
coccus pyogenes  may,  by  means  of  their  products,  produce  areas 
of  softening  in  the  spinal  cord,  and  this  may  furnish  an  explana- 
tion of  some  of  the  lesions  found  clinically.  It  is  also  possible 
that  some  serous  inflammations  may  be  produced  in  the  same 
way. 

B.  Disturbances  of  Metabolism,  etc.  —  It  will  easily  be  real- 
ised that  such  profound  tissue  changes  as  have  been  detailed 
cannot  occur  without  great  interference  with  the  normal  bodily 
metabolism.  General  malnutrition  and  cachexia  are  of  common 
occurrence,  and  it  is  a  striking  fact  found  by  experiment  that 
after  injection  of  bacterial  products,  e.g.  of  the  diphtheria  bacil- 
lus, a  marked  loss  of  body  weight  often  occurs  which  may  be 
progressive,  and  ultimately  lead  to  the  death  of  the  animal.  In 
bacterial  disease  assimilation  is  often  imperfect,  for  the  digestive 
glands  are  affected,  it  may  be,  by  actual  poisoning  by  bacterial 
products,  it  may  be  by  the  occurrence  of  fever.  The  fatty  de- 
generations which  are  so  common  are  indicative  of  a  breaking 
down  of  the  proteid  molecules,  and  are  associated  with  increased 
urea  production,  while  the  degeneration  of  the  kidney  epithe- 
lium renders  the  excretion  of  waste  products  deficient  or  im- 
possible, and  this  is  not  infrequently  the  immediate  cause  of 
death.  But  of  all  the  changes  in  metabolism  the  most  difficult 
to  understand  is  the  occurrence  of  that  interference  with  the 
heat-regulating  mechanism  which  results  in  fever.  The  degree 
and  course  of  the  latter  vary,  sometimes  conforming  to  a  more 
or  less  definite  type,  where  the  bacilli  are  selective  in  their  field 
of  operation,  as  in  croupous  pneumonia  or  typhoid,  sometimes 
being  of  a  very  irregular  kind,  especially  when  the  bacteria 
from  time  to  time  invade  fresh  areas  of  the  body,  as  in  pyaemic 
affections.  The  main  point  of  interest  regarding  the  develop- 
ment of  fever  is  as  to  whether  it  is  a  direct  effect  of  the  circu- 
lation of  bacterial  toxins,  or  if  it  is  to  be  looked  on  as  part  of 


1 70  RELATIONS   OF   BACTERIA  TO   DISEASE. 

the  reaction  of  the  body  against  the  irritant.  This  question 
has  still  to  be  settled,  and  all  that  we  can  do  is  to  adduce 
certain  facts  bearing  on  it.  Thus  in  diphtheria  and  tetanus, 
where  toxic  action  leading  to  degeneration  plays  such  an 
important  part,  fever  may  be  a  very  subsidiary  feature,  except 
in  the  terminal  stage  of  the  latter  disease ;  and  in  fact  in  diph- 
theria profoundly  toxic  effects  may  be  produced  without  the  least 
interference  with  heat  regulation.  On  the  other  hand,  in  bac- 
terial disease,  where  defensive  and  reparative  processes  predom- 
inate, fever  is  rarely  absent,  and  it  is  nearly  always  present  when 
an  active  leucocytosis  is  going  on.  In  this  connection  it  may  be 
remarked  that  several  observers  have  found  that,  when  a  relatively 
small  amount  of  the  dead  bodies  of  certain  bacteria  are  injected 
into  an  animal,  fever  occurs  ;  while  the  injection  of  a  large  amount 
of  the  same  is  followed  by  subnormal  temperatures  and  rapidly 
fatal  collapse.  It  might  appear  as  if  this  indicated  that  the 
occurrence  of  fever  had  a  beneficial  effect,  but  this  is  one  of 
the  points  at  issue.  Certainly  such  an  effect  is  not  due  to  the 
bacteria  being  unable  to  multiply  at  the  higher  degrees  of  tem- 
perature occurring  in  fever,  for  this  has  been  shown  not  to  be  the 
case.  Whether  the  increase  of  bodily  temperature  indicates  the 
occurrence  of  changes,  e.g.  in  the  way  of  the  production  of  bacteri- 
cidal bodies,  etc.,  is  a  question,  and  all  attempts  made  hitherto 
towards  its  solution  have  been  unsuccessful.  On  the  one  hand, 
it  is  stated  that  the  maintaining,  during  an  illness  due  to  bacteria, 
of  an  animal's  body  at  its  normal  temperature  by  means  of  anti- 
pyretics favours  its  recovery ;  and  on  the  other  hand  it  has  been 
found  that  the  placing  of  such  an  animal  in  an  atmosphere  at  a 
temperature  above  its  normal  also  favours  recovery.  It  is  evident 
that  the  experiments  are  not  parallel,  and,  in  fact,  that  from  the 
latter  no  conclusion  can  be  drawn,  as  the  additional  factor  of  the 
condition  of  the  heat-regulating  mechanism,  when  the  bodily 
temperature  rises,  is  also  introduced.  If  we  consider  the  site  of 
the  heat  production  in  fever  we  again  are  in  difficulties.  It  might 
appear  as  if  the  tissue  destruction,  indicated  by  the  occurrence  of 
fatty  degeneration,  would  lead  to  heat  development,  but  frequently 
excessive  heat  production  with  increased  proteid  metabolism 
occurs  without  any  discoverable  changes  in  the  tissues ;  and 
further,  in  phosphorus  poisoning  there  is  little  fever  with  great 
tissue  destruction.  The  increased  work  performed  by  the  heart 


THE   TOXINS   PRODUCED   BY   BACTERIA.  171 

in  most  bacterial  infections  no  doubt  contributes  to  the  rise  of 
bodily  temperature.  But  we  must  bear  in  mind  that  in  fever 
there  is  more  than  mere  increase  of  heat  production  —  there  is 
also  a  diminished  loss  of  heat  from  interference  with  the  nervous 
mechanism  of  the  sweat  apparatus.  The  known  facts  would 
indicate  that  in  fever  there  may  be  a  factor  involving  the  nervous 
system  to  be  taken  into  account.  The  whole  subject  is  thus  very 
obscure. 

Symptoms.  —  Many  of  the  symptoms  occurring  in  bacterial 
affections  are  produced  by  the  histological  changes  mentioned, 
as  can  be  readily  understood  ;  whilst  in  the  case  of  others,  corre- 
sponding changes  have  not  yet  been  discovered.  Of  the  latter, 
those  associated  with  fever,  with  its  disturbances  of  metabolism 
and  manifold  affections  of  the  various  systems,  are  the  most 
important.  The  nervous  system  is  especially  liable  to  be  affected 
—  convulsions,  spasms,  coma,  paralysis,  etc.,  being  common. 
The  symptoms  due  to  disturbance  or  abolition  of  the  functions 
of  secretory  glands  also  constitute  an  important  group,  forming, 
as  they  do,  a  striking  analogy  to  what  is  found  in  the  action  of 
various  drugs. 

These  tissue  changes  and  symptoms  are  given  only  as  illus- 
trative examples,  and  the  list  might  easily  be  greatly  amplified. 
The  important  fact,  however,  is  that  nearly  all,  if  not  quite  all, 
the  changes  found  throughout  the  organs  (without  the  actual  pres- 
ence of  bacteria),  and  also  the  symptoms  occurring  in  infective 
diseases,  can  either  be  experimentally  reproduced  by  the  injection 
of  bacterial  poisons  or  have  an  analogy  in  the  action  of  drugs. 

THE  TOXINS  PRODUCED  BY  BACTERIA. 

Early  Work  on  Toxins.  —  We  know  that  bacteria  are  capable 
of  giving  rise  to  poisonous  bodies  within  the  animal  body  and 
also  in  artificial  media.  As  we  shall  see,  we  know  comparatively 
little  of  the  actual  nature  of  such  bodies,  and  therefore  we  apply 
to  them  as  a  class  the  general  term  toxins.  The  necessity  for 
accounting  for  the  general  pathogenic  effects  of  certain  bacteria, 
which  in  the  corresponding  diseases  were  not  distributed  through- 
out the  whole  body,  directed  attention  to  the  probable  existence 
of  such  toxins ;  and  the  first  to  systematically  study  the  produc- 
tion of  such  poisonous  bodies  was  Brieger.  This  observer 
isolated  from  putrefying  substances,  and  also  from  bacterial 


1/2  RELATIONS    OF   BACTERIA   TO    DISEASE. 

cultures,  nitrogen-containing  bodies,  which  he  called  ptomaines. 
Similar  bodies  occurring  in  the  ordinary  metabolic  processes  of 
the  body  had  previously  been  described  and  called  leucomaines. 
Ptomaines  isolated  from  pathogenic  bacteria  in  no  case,  except 
perhaps  in  tetanus,  reproduced  the  symptoms  of  the  disease. 
The  methods  by  which  they  were  isolated  were  faulty,  and  they 
have  therefore  only  a  historic  interest. 

The  introduction  of  the  principle  of  rendering  fluid  cultures 
bacteria-free  by  filtration  through  unglazed  porcelain,  and  its 
application  by  Roux  and  Yersin  to  obtain,  in  the  case  of  the  B. 
diphtheriae,  a  solution  containing  a  toxin  which  reproduced  the 
symptoms  of  this  disease  (vide  Chapter  XVI.),  encouraged  the 
further  inquiry  as  to  the  nature  of  this  toxin.  The  products  of 
the  B.  diphtheriae  were  investigated  again  by  Brieger,  now  in 
conjunction  with  C.  Fraenkel.  The  method  employed  was  pre- 
cipitation by  alcohol,  and  the  material  obtained  gave  certain 
reactions  of  the  parent  fluids.  This  substance,  if  it  did  not  con- 
sist entirely  of  the  diphtheria  toxin,  certainly  contained  the  latter, 
and  from  resemblances  observed  in  it  to  serum  albumin,  was 
called  by  its  discoverers  a  toxalbumin.  Similar  toxic  bodies 
were  obtained  from  the  bacteria  of  tetanus,  typhoid,  and  cholera, 
and  also  from  the  staphylococcus  aureus,  but  with  these,  though 
death  occurred  from  their  injection,  no  characteristic  symptoms 
or  pathological  effects  were  observed.  They  probably  consisted 
largely  of  albumoses,1  and  contained  the  toxic  bodies  in  mixture 
with  other  substances. 

The  Occurrence  of  Bacterial  Toxins.  —  The  following  may  be 
regarded  as  the  chief  facts  regarding  bacterial  toxins  which  have 

1  In  the  digestion  of  albumins  by  the  gastric  and  pancreatic  juices  the  albumoses 
are  a  group  of  bodies  formed  preliminarily  to  the  elaboration  of  peptone.  Like  the 
latter  they  differ  from  the  albumins  in  their  not  being  coagulated  by  heat,  and  in 
being  slightly  dialysable.  They  differ  from  the  peptones  in  being  precipitated  by 
dilute  acetic  acid  in  presence  of  much  sodium  chloride,  and  also  by  neutral  saturated 
sulphate  of  ammonia.  Both  are  precipitated  by  alcohol.  The  first  albumoses  formed 
in  digestion  are  proto-albumose  and  hetero-albumose,  which  differ  in  the  insolubility 
of  the  latter  in  hot  and  cold  water  (insolubility  and  coagulability  are  quite  different 
properties).  They  have  been  called  the  primary  albumoses.  By  further  digestion 
both  pass  into  the  secondary  albumose,  deutero-albumose,  which  differs  slightly  in 
chemical  reactions  from  the  parent  bodies,  e.g.  it  cannot  be  precipitated  from  watery 
solutions  by  saturated  sodium  chloride  unless  a  trace  of  acetic  acid  be  present.  Dys- 
albumose  is  probably  merely  a  temporary  modification  of  hetero-albumose.  Further 
digestion  of  deutero-albumose  results  in  the  formation  of  peptone. 


INTRACELLULAR   AND   EXTRACELLULAR   TOXINS.       173 

been  revealed  by  the  study,  partly  of  the  bodily  tissues  of 
animals  infected  by  the  bacteria  concerned,  partly  by  what 
occurs  in  artificial  cultures  of  these  bacteria.  The  dead  bodies 
of  certain  species  have  been  found  to  be  very  toxic.  When,  for 
instance,  tubercle  bacilli  are  killed  by  heat  and  injected  into  a 
susceptible  animal  tubercular  nodules  are  found  to  develop  round 
the  sites  where  they  have  lodged.  From  this  it  is  inferred  that 
they  must  have  contained  characteristic  toxins,  seeing  that 
characteristic  lesions  result.  The  bodies  of  the  cholera  vibrio 
are  likewise  toxic.  Such  intracellular  toxins,  as  they  have  been 
called,  may  appear  in  the  fluids  in  which  the  bacteria  are  living 
(i)  by  excretion  in  an  unaltered  or  altered  condition,  (2)  by  the 
disintegration  of  the  bodies  of  the  organisms  which  we  know  are 
always  dying  in  any  bacterial  growth.  Sometimes,  on  the  other 
hand,  the  media  in  which  bacteria  are  growing  become  extremely 
toxic.  This  is  much  greater  in  some  cases  than  in  others.  The 
two  best  examples  of  bacteria  thus  producing  soluble  toxins  are 
the  diphtheria  and  tetanus  bacilli.  In  these  and  similar  cases, 
when  bouillon  cultures  are  filtered  bacterium-free  by  means  of  a 
porcelain  filter,  highly  toxic  fluids  are  obtained,  which  on  injec- 
tion into  animals  reproduce  the  highly  characteristic  symptoms 
of  the  corresponding  diseases.  In  the  case  of  the  B.  anthracis 
and  of  many  others,  at  any  rate  when  growing  in  artificial  media, 
such  toxin  production  is  much  less  marked,  a  filtered  bouillon 
culture  being  relatively  non-toxic.  It  is  probable,  however,  that 
this  may  not  occur  when  the  bacillus  is  growing  in  an  animal 
body,  for  we  have  often  here  well-marked  evidence  of  pathogenic 
effects  being  produced  at  a  distance  from  the  actual  focus  of 
bacterial  growth.  This  is  further  an  instance  of  what  we  have 
strong  reason  to  believe  sometimes  occurs,  namely,  that  the 
toxins  produced  by  bacteria,  when  these  are  growing  in  the  ani- 
mal body,  differ  somewhat  from  the  toxins  produced  by  the 
same  bacteria  growing  in  artificial  media.  Poisons  appearing  in 
cultures  have  been  called  extracellular  toxins,  but,  as  we  shall 
see,  we  cannot  as  yet  say  whether  they  are  excreted  by  the 
bacteria,  or  whether  they  are  produced  by  the  bacteria  acting  on 
the  constituents  of  the  media.  We  therefore  cannot  as  yet  draw 
a  hard  and  fast  line  between  intra-  and  extracellular  toxins,  but 
the  terms  are  convenient,  and  may  apply  to  two  actually 
different  sets  of  bodies.  That  the  poisonous  capacities  of  a 


1/4  RELATIONS    OF   BACTERIA   TO    DISEASE. 

bacterium  may  be  very  complicated  is  shown  by  what  is  known 
in  the  case  of  the  vibrio  cholerae,  where  the  poisons  which  dis- 
solve out  into  the  culture  fluid  are  quite  different  in  their  nature 
from  those  which  act  when  the  dead  bacteria  are  injected  into 
an  animal.  The  extracellular  toxins  are  the  more  easily  obtain- 
able in  large  quantities,  and  it  is  their  nature  and  effects  which 
are  best  known. 

The  Nature  of  Toxins.  —  Nearly  all  of  what  little  is  known 
regarding  this  subject  relates  to  the  extracellular  toxins.  The 
earlier  investigations  upon  toxins  suggested  that  analogies  exist 
between  the  modes  of  bacterial  action  and  what  takes  place  in 
ordinary  gastric  digestion,  and  the  idea  has  been  worked  out  for 
certain  pathogenic  bacteria  by  Sidney  Martin.  This  observer 
took,  not  solutions  artificially  made  up  with  albumoses,  but  the 
natural  fluids  of  the  body  or  definite  solutions -of  albumins,  and, 
further,  never  subjected  the  results  of  the  bacterial  growth  to 
heat  above  40°  C.  or  to  any  stronger  agent  than  absolute 
alcohol.  He  found  that  albumoses  and  sometimes  peptones 
were  formed  by  the  action  of  the  pathogenic  bacteria  studied, 
and  further,  that  the  precipitate  containing  these  albumoses 
was  toxic.  In  certain  cases  the  process  of  splitting  up  of  the 
albumins  went  further  than  in  peptic  digestion,  and  organic 
bases  or  acids  might  be  formed.  According  to  Martin,  the 
characteristic  symptoms  of  the  diseases  could  be  explained  by 
compound  actions,  in  which  the  albumoses  were  responsible 
for  some  of  the  effects,  the  other  bodies  for  others.  A  simi- 
lar digestive  action  has  been  traced  in  the  case  of  the  tubercle 
bacillus  by  Kiihne. 

Further  evidence  that  bacterial  toxins  are  either  albumoses 
or  bodies  having  a  still  smaller  molecule  is  furnished  by  C.  J. 
Martin.  This  worker,  by  filling  the  pores  of  a  Chamberland 
bougie  with  gelatin,  has  obtained  what  is  practically  a  strongly 
supported  colloid  membrane  through  which  dialysis  can  be  made 
to  take  place  under  the  great  pressure  say  of  compressed  oxy- 
gen. He  finds  that  in  such  an  apparatus  toxins,  at  least  the  two 
kinds  tried,  will  pass  through  just  as  an  albumose  will. 

Brieger  and  Boer,  working  with  bouillon  cultures  of  diphtheria 
and  tetanus,  have,  by  precipitation  with  zinc  chloride,  separated 
bodies  which  show  characteristic  toxic  properties,  but  which  have 
the  reactions  neither  of  peptone,  albumose,  nor  albuminate,  and 


THE   NATURE   OF   TOXINS.  175 

the  nature  of  which  is  unknown.  It  has  also  been  found  that  the 
bacteria  of  tubercle,  tetanus,  diphtheria,  and  cholera  can  pro- 
duce toxins  when  growing  in  proteid-free  fluids.  In  the  case  of 
diphtheria,  when  the  toxin  is  produced  in  such  a  fluid  a  proteid 
reaction  appears.  Of  course  this  need  not  necessarily  be 
caused  by  the  toxin.  Further  investigation  is  here  required,  for 
Uschinsky,  applying  Brieger  and  Boer's  method  to  a  toxin  so 
produced,  states  that  the  toxin  body  is  not  precipitated  by  zinc 
salts,  but  remains  free  in  the  medium.  If  the  toxins  are  really 
non-proteid  they  may,  on  the  one  hand,  be  the  final  product  of 
a  digestive  action,  or  they  may  be  the  manifestation  of  a  separate 
vital  activity  on  the  part  of  the  bacteria.  On  the  latter  theory 
the  toxicity  of  the  toxalbumins  of  Brieger  and  Fraenkel,  and  of 
the  toxic  albumoses  of  Martin,  may  be  due  to  the  precipitation 
of  the  true  toxins  along  with  these  other  bodies.  From  the 
chemical  standpoint  this  is  quite  possible.  When  we  take  into 
account  the  extraordinary  potency  of  these  poisons  (in  the  case 
of  tetanus  the  fatal  dose  of  the  pure  poison,  for  a  guinea-pig 
must  often  be  less  than  .00000 1  gr.),  we  must  realise  that  all 
attempts  by  present  chemical  methods  to  isolate  them  in  a  pure 
condition  are  not  likely  to  be  successful,  and  of  their  real  nature 
we  know  nothing.  Amongst  the  properties  of  the  extracellular 
toxins,  however,  are  the  following :  They  are  certainly  all  un- 
crystallisable ;  they  are  soluble  in  water  and  they  are  dialysable  ; 
they  are  precipitated  along  with  proteids  by  concentrated  alcohol, 
and  also  by  ammonium  sulphate ;  if  they  are  proteids  they  are 
either  albumoses  or  allied  to  the  albumoses  ;  they  are  relatively  un- 
stable, having  their  toxicity  diminished  or  destroyed  by  heat  (the 
degree  of  heat  which  is  destructive  varies  much  in  different  cases), 
light,  and  by  certain  chemical  agents.  Their  potency  is  often 
altered  in  the  precipitations  practised  to  obtain  them  in  a  pure 
or  concentrated  condition,  but  among  the  precipitants  ammonium 
sulphate  has  little  if  any  harmful  effect.  Regarding  the  toxins 
which  are  more  intimately  associated  with  the  bacterial  cell  we 
know  much  less,  but  it  is  probable  that  their  nature  is  similar, 
though  some  of  them  at  least  are  not  so  easily  injured  by 
heat,  e.g.  in  the  case  of  the  tubercle  bacilli  already  mentioned. 
In  the  case  of  all  toxins  the  fatal  dose  for  an  animal  varies 
directly  with  the  species,  body  weight,  age,  and  previous  con- 
ditions as  to,  e.g.,  food,  temperature,  etc.  In  estimating  the 


i;6  RELATIONS   OF   BACTERIA  TO   DISEASE. 

minimal  lethal  dose  of  a  toxin  these  factors  must  be  carefully 
considered. 

The  following  is  the  best  method  of  obtaining  concentrated  extracellular 
toxins.  The  toxic  fluid  is  placed  in  a  shallow  dish,  and  ammonium  sulphate 
crystals  are  well  stirred  in  till  no  more  dissolve.  Fresh  crystals  to  form  a 
bulk  nearly  equal  to  that  of  the  whole  fluid  are  added,  and  the  dish  set  in  an 
incubator  at  37°  C.  over  night.  Next  day  a  brown  scum  of  precipitate  will  be 
found  floating  on  the  surface.  This  contains  the  toxin.  It  is  skimmed  off 
with  a  spoon,  placed  in  watch-glasses,  and  these  are  dried  in  vacuo  and  stored 
in  the  dark,  also  in  vacua,  or  in  an  exsiccator  containing  strong  sulphuric  acid. 
For  use  the  contents  of  one  are  dissolved  up  in  a  little  normal  saline  solution. 

The  comparison  of  the  action  of  bacteria  in  the  tissues  in  the 
production  of  these  toxins  to  what  takes  place  in  the  gastric 
digestion,  has  raised  the  question  of  the  possibility  of  the  elabo- 
ration by  these  bacteria  of  ferments  by  which  the  process  may 
be  started.  The  problem  of  toxin  formation  is  thus  complicated. 
Sidney  Martin  has  described  toxic  albumoses  as  occurring  in  all 
the  diseases  he  investigated,  viz.,  anthrax,  ulcerative  endocarditis, 
diphtheria,  and  tetanus.  In  each  of  these  cases,  therefore,  we 
would  be  led  to  suppose  that  ferments  might  be  produced, 
which  we  may  look  on  as  the  primary  toxic  agent  which  acts  by 
digesting  surrounding  material  and  producing  albumoses  which 
form  the  secondary  poisons.  From  the  standpoint  of  the  bac- 
terium this  process  would  simply  be  a  preparation  of  food  for 
further  intracellular  digestion.  Hitherto  all  attempts  at  the  isola- 
tion of  bacterial  ferments  of  such  a  nature  have  failed. 

The  question  of  fermentation  has  been  chiefly  discussed  with 
regard  to  what  happens  in  diphtheria  and  tetanus.  Apart  from 
the  fact  that  a  digestive  action  has  occurred,  which  the  presence 
of  albumoses  in  the  body  of  an  animal  dead  of  these  diseases 
affords,  the  chief  available  evidence  for  the  existence  of  ferments 
lies  in  this,  that  the  toxic  products  of  the  bacteria  involved  lose 
their  toxicity  by  exposure  to  a  temperature  which  puts  an  end  to 
the  diastatic  activity  of  such  an  undoubted  ferment  as  that  of  the 
gastric  juice.  If  a  bouillon  containing  diphtheria  toxin  be  heated 
at  65°  C.  for  one  hour,  it  is  found  to  have  lost  much  of  its  toxic 
effect,  and  in  the  case  of  B.  tetani  all  the  toxicity  is  lost  by  ex- 
posure at  this  temperature.  In  both  diseases  there  is  a  still 
further  fact  which  is  adduced  in  favour  of  a  ferment  being  con- 
cerned in  the  toxic  action,  namely,  the  existence  of  a  definite 


PROPERTIES    OF   BACTERIAL   FERMENTS.  177 

period  of  incubation  between  the  injection  of  the  toxic  bodies 
and  the  appearance  of  symptoms.  This  may  be  interpreted  as 
showing  that  after  the  introduction  of  say  a  filtered  bouillon 
culture,  further  chemical  substances  are  formed  in  the  body  be- 
fore the  actual  toxic  effect  is  produced.  In  the  case  of  tetanus 
at  least  the  delay,  however,  may  be  explained  by  the  fact  that 
the  poison  apparently  has  to  travel  up  the  nerve  trunks  before 
the  real  poisonous  action  is  developed.  With  some  poisons  pres- 
ently to  be  mentioned  which  are  closely  allied  to  the  bacterial 
toxins  an  incubation  period  may  not  exist.  It  would  not  be 
prudent  to  dogmatise  as  to  whether  the  toxins  do  or  do  not  be- 
long to  such  an  ill-defined  group  of  substances  as  the  ferments. 
It  may  be  pointed  out,  however,  that  the  essential  concept  of  a 
ferment  is  that  of  a  body  which  can  originate  change  without 
itself  being  changed,  and  no  evidence  has  been  adduced  that 
toxins  fulfil  this  condition.  Another  property  of  ferments  is 
that,  so  long  as  the  products  of  fermentation  are  removed,  the 
action  of  a  given  amount  of  ferment  is  indefinite.  Again,  in  the 
case  of  toxins  no  evidence  of  such  an  occurrence  has  been  found. 
A  certain  amount  of  a  toxin  is  always  associated  with  a  given 
amount  of  disease  effect,  though  a  process  of  elimination  of  waste 
products  must  be  all  the  time  going  on  in  the  animal's  body. 
Again,  too  much  importance  must  not  be  attached  to  loss  of 
toxicity  by  toxins  at  relatively  low  temperatures.  Many  proteids 
show  a  tendency  to  change  at  such  temperatures ;  for  instance, 
if  egg  albumin  be  kept  long  enough  at  55°  C.  nearly  the  whole 
of  it  will  be  coagulated.  Such  considerations  suggest  that  the 
relation  of  toxic  action  to  fermentation  must  be  left  an  open 
question. 

Similar  Vegetable  and  Animal  Poisons.  —  Within  recent  years  it  has  been 
found  that  the  bacterial  poisons  belong  to  a  group  of  toxic  bodies  all  present- 
ing very  similar  properties,  other  members  of  which  occur  widely  in  the  vege- 
table and  animal  kingdoms.  Among  plants  the  best-known  examples  are  the 
ricin  and  abrin  poisons,  obtained  by  making  watery  emulsions  of  the  seeds 
of  the  Ricinus  communis  and  the  Abrus  precatorius  (jequirity)  respectively. 
From  the  Robinia  pseudacacia  another  poison — robin  —  belonging  to  the  same 
group  is  obtained.  The  chemical  reactions  of  ricin  and  abrin  correspond  to 
those  of  the  bacterial  toxins.  They  are  soluble  in  water,  they  are  precipitable 
by  alcohol,  but  being  less  easily  dialysable  than  the  albumoses  they  have  been 
called  toxalbumins.  Their  toxicity  is  seriously  impaired  by  boiling,  and  they 
also  gradually  become  less  toxic  on  being  kept.  Both  are  among  the  most 
powerful  poisons  known  — ricin  being  the  more  fatal.  When  they  are  in- 


i;8  RELATIONS   OF   BACTERIA   TO   DISEASE. 

jected  subcutaneously  a  period  of  twenty-four  hours  usually  elapses  —  whatever 
be  the  dose  —  before  symptoms  set  in.  Both  tend  to  produce  great  inflamma- 
tion at  the  seat  of  inoculation,  which  in  the  case  of  ricin  may  end  in  an  acute 
necrosis ;  in  fatal  cases  haemorrhagic  enteritis  and  nephritis  may  be  found. 
Both  act  as  irritants  to  mucous  membranes,  abrin  especially  being  capable  of 
setting  up  most  acute  conjunctivitis. 

It  is  also  certain  that  the  poisons  of  scorpions  and  of  poisonous  snakes 
belong  to  the  same  group.  The  poisons  derived  from  the  latter  are  usually 
called  venins,  and  a  very  representative  group  of  such  venins  derived  from 
different  species  has  been  studied.  To  speak  generally  there  is  derivable 
from  the  natural  secretions  of  the  poison  glands  a  series  of  venins  which  have 
all  the  reactions  of  the  bodies  previously  considered.  Like  ricin  and  abrin, 
they  are  not  so  easily  dialysable  as  bacterial  toxins,  and  therefore  have  also 
been  classed  as  toxalbumins.  Their  properties  are  also  similar ;  many  of 
them  are  destroyed  by  heat,  but  the  degree  necessary  here  also  varies  much. 
There  is  also  evidence  that  in  a  crude  venin  there  may  be  several  poisons 
differently  sensitive  to  heat.  All  the  venins  are  very  powerful  poisons,  but 
here  there  is  practically  no  period  of  incubation  —  the  effects  are  almost  im- 
mediate. The  toxicity  of  the  venins  varies  much  with  the  animal  employed, 
but  chiefly  with  the  species  of  snake  from  which  it  was  derived.  For  instance, 
.47  milligramme  of  crude  venin  from  the  Indian  cobra  will  kill  a  rabbit  in 
three  or  four  hours.  In  the  case  of  the  American  rattlesnake  the  dose  would 
be  3.5  milligrammes,  and  in  that  of  the  Australian  hoplocephalus  variegatus 
2.5  milligrammes.  The  general  effects  of  these  vary  with  the  dose,  and  slight 
variations  also  exist  between  the  effects  of  venins  of  different  snakes.  Thus 
cobra  poison  is  said  to  produce  rapid  paralysis  of  the  lips,  tongue,  larynx,  and 
respiratory  apparatus,  from  which  death  results.  On  the  other  hand,  the  venin 
of  the  daboia  of  Ceylon  is  said  to  cause  violent  general  convulsions,  succeeded 
by  paralysis,  but  with  very  little  respiratory  affection.  In  the  case  of  a  dose 
not  sufficient  to  cause  immediate  death  from  its  general  effects,  often  the  most 
acute  and  widespread  necrosis  may  occur  in  a  few  hours  round  the  site  of 
inoculation. 

The  Theory  of  Toxic  Action.  —  While  we  know  little  of  the 
chemical  nature  of  any  toxins,  we  may,  from  our  knowledge  of 
their  properties,  group  together  the  tetanus  and  diphtheria 
poisons,  ricin,  abrin,  snake  poisons,  and  scorpion  poisons. 
Besides  the  points  of  agreement  already  noted,  all  possess  the 
further  property  that,  as  will  be  afterwards  described,  when 
introduced  into  the  bodies  of  susceptible  animals  they  stimulate 
the  production  of  substances  called  antitoxins.  The  nature  of 
the  antagonism  between  toxin  and  antitoxin  will  be  discussed 
later.  Here,  to  explain  what  follows,  it  may  be  stated  (i)  that  the 
molecule  of  toxin  most  probably  forms  a  chemical  combination 
with  the  molecule  of  antitoxin,  and  (2)  that  it  has  been  shown 
that  toxin  molecules  may  lose  much  of  their  toxic  power  and 


THE   THEORY   OF   TOXIC   ACTION.  179 

still  be  capable  of  uniting  with  exactly  the  same  proportion  of 
antitoxin  molecules.  From  these  and  other  circumstances, 
Ehrlich  has  advanced  the  view  that  the  toxin  molecule  has  a 
very  complicated  structure,  and  contains  two  atom-groups.  One 
of  these,  the  haptophorous  (aTrreiv,  to  bind  to),  is  that  by  which 
combination  takes  place  with  the  antitoxin  molecule  and  also 
with  presumably  corresponding  molecules  naturally  existing  in 
the  tissues.  The  other  atom-group  he  calls  the  toxophorous,  and 
it  is  to  this  that  the  toxic  effects  are  due.  This  atom-group  is 
bound  to  the  cell  elements,  e.g.  the  nerve  cells  in  tetanus,  by  the 
haptophorous  group.  Ehrlich  holds  that  the  toxophorous  group 
is  the  more  complicated  and  also  the  less  stable.  It  is  known 
that,  for  instance,  a  diphtheria  toxin  obtained  by  the  filtration  of 
a  bouillon  culture  loses  its  toxicity  when  subjected  to  such 
physical  agencies  as  light  and  heat,  and  to  certain  chemical 
substances.  Ehrlich  explains  this  on  the  theory  that  the  toxo- 
phorous group  undergoes  disintegration.  And  if  we  suppose 
that  the  haptophorous  group  remains  unaffected,  we  can  then 
understand  how  a  toxin  may  have  its  toxicity  diminished  and 
still  require  the  same  proportion  of  antitoxin  molecules  for  its 
neutralisation.  To  the  bodies  whose  toxophorous  atom-groups 
have  become  degenerated,  Ehrlich  gives  the  name  toxoids,  and 
more  recently  he  has  called  them  toxones.  He  states  that  he 
has  found  evidence  that  similar  bodies  may  be  directly  formed 
by  the  diphtheria  bacillus  and  not  as  the  result  of  subsequent 
degeneration.  Such  observations  are  of  importance,  not  only 
as  .throwing  light  on  the  constitution  of  the  toxin  molecule,  but 
also  as  affording  an  explanation  of  how  altered  toxins  (toxoids) 
can  act  as  immunising  agents  by  stimulating  antitoxin  formation. 
The  theory  may  also  afford  an  explanation  of  what  has  been 
suspected,  namely,  that  in  some  instances  toxins  derived  from 
different  sources  may  be  related  to  one  another.  For  example, 
Ehrlich  has  pointed  out  that  ricin  produces  in  a  susceptible 
animal  body  an  antitoxin  which  corresponds  almost  completely 
with  that  produced  by  another  vegetable  poison,  robin  (vide  supra), 
though  ricin  and  robin  are  certainly  different.  This  may  be  due 
to  the  fact  that  robin  is  a  toxoid  of  ricin,  i.e.  their  haptophorous 
groups  correspond,  while  their  toxophorous  differ.  The  evidence 
on  which  Ehrlich's  deductions  are  based  is  of  a  very  weighty  char- 
acter, and  will  be  again  referred  to  in  the  chapter  on  Immunity. 


CHAPTER    VII. 
INFLAMMATORY   AND   SUPPURATIVE   CONDITIONS. 

THIS  subject  is  an  exceedingly  wide  one,  and  embraces  a  great 
many  pathological  conditions  which  in  their  general  characters 
and  results  are  widely  different.  Thus  in  addition  to  suppura- 
tion, various  inflammations,  ulcerative  endocarditis,  septicaemia, 
and  pyaemia,  will  come  up  for  consideration.  With  regard  to 
these  the  two  following  general  statements,  established  by  bac- 
teriological research,  may  be  made  in  introducing  the  subject. 
In  the  first  place,  there  is  no  one  specific  organism  for  any  one 
of  these  conditions ;  various  organisms  may  produce  them,  and 
not  infrequently  more  than  one  organism  may  be  present  to- 
gether. In  the  second  place,  the  same  organism  may  produce 
widely  varying  results  under  different  circumstances,  —  at  one 
time  a  local  inflammation  or  abscess,  at  another  multiple  suppu- 
rations or  a  general  septicaemia.  The  principles  on  which  this 
diversity  in  results  depends  have  already  been  explained  (p.*  158). 

It  may  be  well  to  emphasise  some  of  the  chief  points  in  the 
pathology  of  these  conditions.  In  suppuration  the  two  main 
phenomena  are  —  (a)  a  progressive  immigration  of  leucocytes, 
chiefly  of  the  polymorphic-nuclear  (neutrophile)  variety,  and  (b) 
a  liquefaction  or  digestion  of  the  supporting  elements  of  the 
tissue  along  with  necrosis  of  the  cells  of  the  part.  The  result 
is  that  the  tissue  affected  becomes  replaced  by  the  cream-like 
fluid  called  pus.  A  suppurative  inflammation  is  thus  to  be 
distinguished  on  the  one  hand  from  an  inflammation  without 
destruction  of  tissue,  and  on  the  other  from  necrosis  or  death 
en  masse,  where  the  tissue  is  not  liquefied,  and  leucocyte 
accumulation  may  be  slight.  When,  however,  suppuration  is 
taking  place  in  a  very  dense  fibrous  tissue,  liquefaction  may  be 
incomplete,  and  a  portion  of  dead  tissue  or  slough  may  remain 
in  the  centre,  as  is  the  case  in  boils.  In  the  case  of  suppuration 
in  a  serous  cavity  the  two  chief  factors  are  the  progressive 

1 80 


SEPTICAEMIA  AND   PY^MIA.  181 

leucocytic  accumulation   and    the  disappearance  of    any  fibrin 
which  may  be  present. 

The  liquefaction  of  the  formed  tissue  elements  in  suppura- 
tion depends  chiefly  upon  a  peptonising  action  of  the  organisms 
or  of  ferments  produced  by  them,  and  the  progressive  leucocytic 
aggregation  is  most  probably  the  effect  of  microbic  products 
which  attract  the  leucocytes,  or  in  other  words  exert  a  positive 
chemiotaxis.  From  this  it  might  be  inferred  that  suppuration  is 
almost  exclusively  related  to  the  presence  of  living  organisms, 
and  this  is  found  to  be  actually  the  case.  Many  experiments 
have  been  performed  to  determine  whether  suppuration  can  be 
produced  by  various  chemical  substances,  such  as  croton  oil,s 
nitrate  of  silver,  turpentine,  etc.,  care,  of  course,  being  taken  to 
ensure  the  absence  of  bacteria.  The  general  result  obtained  by 
independent  observers  is  that  as  a  rule  suppuration  does  not 
follow,  but  that  in  certain  animals  and  with  certain  substances 
it  may,  the  pus  being  free  from  bacteria.  It  is  still,  however, 
questioned  by  some  whether  the  pus  thus  produced  really  cor- 
responds histologically  and  chemically  with  that  due  to  bacterial 
action.  Buchner  showed  that  suppuration  may  be  produced  by 
the  injection  of  dead  bacteria,  e.g.  sterilised  cultures  of  bacillus 
pyocyaneus,  etc.  The  subject  has  now  more  a  scientific  than  a 
practical  interest,  and  the  general  statement  may  be  made  that 
practically  all  cases  of  true  suppuration  met  with  clinically  are* 
due  to  the  'action  of  living  micro-organisms. 

The  term  septicamia  is  applied  to  conditions  in  which  the 
organisms  multiply  within  the  blood  and  give  rise  to  symptoms 
of  general  poisoning,  without,  however,  producing  abscesses  in 
the  organs.  In  all  cases  of  septicaemia  the  organisms  are  more 
numerous  in  the  capillaries  of  internal  organs  than  in  the  peri- 
pheral circulation,  and,  in  the  case  of  the  human  subject,  it  is 
often  impossible  to  detect  any  in  the  blood  during  life,  though 
they  may  be  seen  in  .large  numbers  in  the  capillaries  of  the 
kidneys,  liver,  etc.,  post  mortem.  The  essential  fact  in  pycemia, 
on  the  other  hand,  is  the  occurrence  of  multiple  abscesses  in  in- 
ternal organs  and  other  parts  of  the  body.  In  most  of  the  cases 
of  typical  pyaemia,  common  in  pre-antiseptic  days,  the  starting- 
point  of  the  disease  was  a  septic  wound  with  bacterial  invasion 
of  a  vein,  leading  to  thrombosis  and  secondary  embolism.  Mul- 
tiple foci  of  suppuration  may  be  produced,  however,  in  other 


\. 


1 82     INFLAMMATORY   AND   SUPPURATIVE   CONDITIONS. 

ways,  as  will  be  described  below  (p.  196).  If  the  term  pycemia 
be  used  to  embrace  all  such  conditions,  their  method  of  produc- 
tion should  always  be  distinguished. 

BACTERIA  AS  CAUSES  OF  INFLAMMATION  AND  SUPPURATION. 

A  considerable  number  of  species  of  bacteria  have  been 
found  in  acute  inflammatory  and  suppurative  conditions,  and  of 
these  many  have  been  proved  to  be  causally  related,  whilst  of 
some  others  the  exact  action  has  not  yet  been  fully  determined. 

Ogston,  who  was  one  of  the  first  to  study  this  question  (in 
1881),  found  that  the  organisms  most  frequently  present  were 
micrococci,  of  which  some  were  arranged  irregularly  in  clusters 
(staphylococci),  whilst  others  formed  chains  (streptococci).  He 
found  that  the  former  were  more  common  in  circumscribed  acute 
abscesses,  the  latter  in  spreading  suppurative  conditions.  Rosen- 
bach  shortly  afterwards  ( 1 884),  by  means  of  cultures,  differentiated 
several  varieties  of  micrococci,  to  which  he  gave  the  following 
special  names :  staphylococcus  pyogenes  aureus,  staphylococcus 
pyogenes  albus,  streptococcus  pyogenes,  micrococcus  pyogenes  tennis. 
Other  organisms  have  been  met  with  in  suppuration,  such  as 
staphylococctis  epidermidis  albus  (Welch),  staphylococcus  pyogenes 
citreus,  stapJiylococcus  cereus  albus,  staphylococcus  cereus  flavus, 
bacillus  pyogenes  foetidus  (Passet),  bacillus  coli  communis,  bacillus 
lactis  aerogencs,  bacillus  aerogenes  capsulatus,  bacillus  pyocyaneus, 
micrococcus  tetragenus,  pneumococcus,  pneumobacillus,  diplococcus 
intracellularis  meningitidis,  and  others. 

In  secondary  inflammations  and  suppurations  following  acute 
diseases  the  corresponding  organisms  have  been  found  in  some 
cases,  such  as  gonococcus,  pneumococcus  of  Fraenkel,  pneumo- 
bacillus  of  Friedlander,  aaH  the  typhoid  bacillus. 

Suppuration  is  also  ^produced  by  the  actinomyces  and  the 
glanders  bacillus,  and  sometimes  chronic  tubercular  lesions  have 
a  suppurative  character. 

Staphylococcus  pyogenes  aureus.  —  Microscopical  Characters. 
—  This  organism  is  a  spherical  coccus  about  .9  /JL  in  diameter, 
which  grows  irregularly  in  clusters  or  masses  (Fig.  65),  and 
occasionally  short  chains  of  4  to  10  units  may  be  seen,  but  may 
readily  be  distinguished  from  short  chains  of  Streptococcus  by 
the  fact  that  the  lines  of  division  between  cocci  lie  parallel  with 


STAPHYLOCOCCUS  PYOGENES  AUREUS. 


183 


the  long  axis  of  the  chain.     It  stains  readily  with  all  the  basic 

aniline  dyes,  and  retains  the 

colour  in  Gram's  method.  '   * 

Cultivation.  —  It  grows 
readily  in  all  the  ordinary 
media  at  the  room  tempera- 
ture, though  much  more 
rapidly  at  the  temperature 

of  the  body.     In  stab-cultures       *-»_  7          **    ^^m 

in  peptone  gelatin  a  streak  of 
growth  is  visible  on  the  day 
after  inoculation,  and  on  the 
second  or  third  day,  liquefac-  „**  •** 

tion   commences  at   the  top. 

.  FIG.  65.  —  Staphylococcus  pyogenes  aureus, 

AS   liquefaction   proceeds,  the     young  culture  on  agar,  showing  clumps  of  cocci. 


growth  falls  to  the  bottom  as.   Stained  with  weak  carbol-fuchsin- 

a  flocculent  deposit,  which  soon  assumes  a  bright  yellow  colour, 
while  a  yellowish  film  may  form  on  the 
surface,  the  fluid  portion  still  remaining 
turbid.  Ultimately  liquefaction  extends 
out  to  the  wall  of  the  tube  (Fig.  66). 
In  gelatin  plates  colonies  may  be  seen 
with  the  low  power  of  the  microscope  in 
twenty-four  hours,  as  little  balls  somewhat 
H  JtagiMJijpl  granular  on  the  surface  and  of  brownish 

^^^  .^,  colour.  On  the  second  day  they  are  visi- 

ble to  the  naked  eye  as  whitish  yellow 
points,  which  afterwards  become  more 
distinctly  yellow.  Liquefaction  occurs 
around  these,  and  little  cups  are  formed, 
at  the  bottom  of  which  colonies  form 
little  yellowish  masses.  On  agar,  a  stroke- 
culture  forms  a  line  of  abundant  yellowish 
growth,  with  smooth,  shining  surface,  well 
formed  after  twenty-four  hours  at  37°  C. 
FIG. 66.— TWO  stab-cui-  Later  it  becomes  bright  orange  in  colour, 

tures  of  staphyiococcus  Pyo-      anc}  resembles  a  streak  of  oil  paint.    Single 

genes  aureus  in  gelatin,  (a) 

10  days  old,  (b)  3  weeks  old :      colonies  on  the  surface  of  agar  are  circu- 
showing  liquefaction  of  the      lar  djscs  of  simiiar  appearance,  which  may 

medium  and   characters   of 

growth.    Natural  size.  reach  2  mm.  or  more  in  diameter.     On 


1 84     INFLAMMATORY   AND    SUPPURATIVE    CONDITIONS. 

potatoes  it  grows  well  at  ordinary  temperature,  forming  a  some- 
what abundant  layer  of  orange  colour.  In  bouillon  it  produces 
a  uniform  turbidity,  which  afterwards  settles  to  the  bottom  as 
an  abundant  layer,  which  assumes  a  brownish-yellow  tint.  In 
the  various  media  it  renders  the  reaction  acid,  and  it  coagulates 
but  does  not  peptonise  milk,  in  which  it  readily  grows.  The 
cultures  have  a  somewhat  sour  odour. 

It  has  considerable  tenacity  of  life  outside  the  body,  cultures 
in  gelatin  often  being  alive  after  having  been  kept  for  several 
months.  It  also  requires  a  rather  higher  temperature  to  kill  it 
than  most  spore-free  bacteria,  viz.  80°  C.  for  half  an  hour 
(Liibbert). 

The  staphylococcus  pyogenes  albus  is  similar  in  character, 
with  the  exception  that  its  growth  on  all  the  media  is  white.  The 
colour  of  the  staphylococcus  aureus  may  become  less  distinctly 
yellow  after  being  kept  for  some  time  in  culture,  but  it  rarely 
assumes  the  white  colour  of  the  staphylococcus  albus,  and  it  has 
not  been  found  possible  to  transform  the  one  organism  into 
the  other. 

Staphylococcus  epidermidis  albus  (Welch)  is,  according  to  its  disco verer,. 
the  most  common  organism  inhabiting  the  skin,  occurring  not  only  on  its  super- 
ficial parts,  but  deeper  down,  in  the  hair  follicles,  and  the  ducts  of  the  seba- 
ceous and  sweat  glands,  where  it  is  not  effected  by  the  most  efficient  methods 
of  disinfection.  Welch  regards  it  as  being  a  "sport1'  of  staphylococcus  py- 
ogenes albus  and  possessed  of  weak  pathogenic  powers.  It  is  the  usual  cause 
of  u  stitch  abscesses,"  following  upon  operative  measures.  Culturally  it  shows 
no  variations  from  the  phenomena  observed  with  staphylococcus  pyogenes 
albus,  excepting  that  it  requires  a  relatively  longer  period  of  time  to  accomplish 
the  same.  It  is  probably  an  attenuated  variety  of  the  staphylococcus  albus. 

The  staphylococcus  pyogenes  citreus,  which  is  less  frequently 
met  with,  differs  in  the  colours  of  the  cultures  being  a  lemon 
yellow,  and  is  less  virulent  than  the  other  two. 

The  staphylococcus  cereus  albus  and  staphylococcus  cereus 
flavus  are  of  much  less  importance.  They  produce  a  wax-like 
growth  on  gelatin  without  liquefaction;  hence  their  name. 

Streptococcus  pyogenes.  —  This  organism  is  a  coccus  of 
slightly  larger  size  than  the  staphylococcus  aureus,  about  I  p  in 
diameter,  and  forms  chains  which  may  contain  a  large  number  of 
members,  especially  when  it  is  growing  in  fluids  (Fig.  67).  The 
chains  vary  somewhat  in  length  in  different  specimens,  and  on 
this  ground  varieties  have  been  distinguished,  e.g.  the  strepto- 


STREPTOCOCCUS   PYOGENES 


I85 


coccus  brevis  and  streptococcus  longus  (vide  infra}.  As  division 
may  take  place  in  many  of  the  cocci  in  a  chain  at  the  same  time, 
the  appearance  of  a  chain  of 
diplococci  is  often  met  with,  the 
line  of  division  lying  always  at 
right  angles  to  the  long  axis  of 
the  chain.  In  young  cultures 
the  cocci  are  fairly  uniform  in 
size,  but  after  a  time  their  size 
presents  considerable  varia- 
tions, many  swelling  up  to  twice 
their  normal  diameter.  These 
are  to  be  regarded  as  involution 
forms.  In  its  staining  reactions 
the  streptococcus  resembles  the 

Staphylococci    described,    being        ^.67.- Streptococcus  pyogenes;  young 

r    J  &     culture  on  agar,  showing  chains  of  cocci, 

readily      Coloured      by      Gram's        Stained  with  weak  carbol-fuchsin.     xiooo. 

method. 

Cultivation.  —  In  cultures  outside  the  body  the  streptococcus 
pyogenes  grows  much  more  slowly  than  the  Staphylococci,  and 
also  dies  out  more  readily,  being  in  every 
respect  a  more  delicate  organism. 

In  peptone  gelatin  a  stab-culture 
shows,  about  the  second  day,  a  thin  line 
which  in  its  subsequent  growth  is  seen 
to  be  formed  of  a  row  of  minute  rounded 
colonies  of  whitish  colour,  which  may  be 
separate  at  the  lower  part  of  the  punc- 
ture. They  do  not  usually  exceed  the 
size  of  a  small  pin's  head,  this  size  being 
reached  about  the  fifth  or  sixth  day.  The 
growth  does  not  spread  on  the  surface 
and  no  liquefaction  of  the  medium  occurs. 
The  colonies  in  gelatin  plates  have  a 
corresponding  appearance,  being  minute 
spherical  points  of  whitish  colour.  On 
the  agar  media  growth  takes  place  along 
the  stroke  as  a  collection  of  small  cir- 
cular discs  of  semi-translucent  appearance,  which  show  a  great 
tendency  to  remain  separate  (Fig.  68).  The  separate  colonies 


FIG.  68.  —  Culture  of  the 
streptococcus  pyogenes  on 
an  agar  plate,  showing 
numerous  colonies  —  three 
successive  strokes.  Twenty- 
four  hours'  growth.  Natural 
size. 


1 86     INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 

remain  small,  rarely  exceeding  i  mm.  in  diameter.  Cultures 
on  agar  kept  at  the  body  temperature  may  often  be  found  to 
be  dead  after  ten  days.  On  potato,  as  a  rule,  no  visible  growth 
takes  place.  In  bouillon,  growth  forms  numerous  minute  gran- 
ules which  afterwards  fall  to  the  bottom,  the  deposit,  which  is 
usually  not  very  abundant,  having  a  sandy  appearance.  The 
appearance  in  broth,  however,  presents  variations  which  have 
been  used  as  an  aid  to  distinguish  different  species  of  strep- 
tococci. It  has  been  found  that  those  which  form  the  longest 
chains  grow  most  distinctly  in  the  form  of  spherical  granules, 
those  forming  short  chains  giving  rise  to  a  finer  deposit.  To 
a  variety  which  forms  distinct  spherules  of  minute  size  the  term 
streptococcus  conglomeratus  has  been  given.  The  question  as  to 
the  existence  of  varieties  of  streptococcus  pyogenes  will  be  dis- 
cussed below. 

Bacillus  coli  communis.  —  The  microscopic  and  cultural  characters  are 
described  in  the  chapter  on  typhoid  fever.  The  bacillus  lactis  aerogenes  and 
the  bacillus  pyogenes  foetidus  closely  resemble  it ;  they  are  either  varieties  or 
closely  related  species.  The  former  is  distinguished  morphologically  by  its 
general  coccus  or  diplococcus  form,  and  culturally  by  its  growth  on  gelatin, 
etc.,  by  producing  more  abundant  gas  formation  in  sugar  media  and  in  acting 
upon  the  starch  of  potato  with  gas  production.  In  milk  cultures  its  coagulative 
action  is  more  rapid,  and  usually  in  this  medium  exhibits  encapsulation.  In 
gelatin  its  growth  is  more  luxuriant  and  whiter  than  that  of  B.  coli. 

Bacillus  aerogenes  capsulatus  sometimes  invades  the  tissues  before  death, 

and  is  characterised  by  the  formation 

,  f  /^w^t*    '    v  of  bubbles  of  gas  in  the  infected  parts. 

*»  *tr  *i  *    **         V  *    '  *ts  cnaracters  are  described  in  Chap- 

^    ^',*%      *  terXVII. 

^ArfVH*      *f  ^wi        *     *  Bacillus  pyocyaneus. — This  or- 

«T ,      <0        j,      "**  \     „"*    .    **     *         ganism  occurs  in  the  form  of  minute 
'    '%£  '    *  .  **N     »f*     rods  i  .5  to  3  /x  in  length  and  less  than 

I  '  »  •*«**.  i*     ,,5  u  in  thickness  (Fig.  69).     Occa- 

%  «    j  V         O  7X 

t  «t         •  .   4  '  .1  »  Vj%J  sionally  two   or  three  are  found  at- 

*f"         "•  ^*      *    •„    -»          *•*/  tached  end  to  end.    They  are  actively 

*****       **,  >    **  ?'  motile,  and  do  not  form  spores.    They 

-     '  %  v     3>*^    „  ~  V  stain  readily  with  the  ordinary  basic 

\       „          *  1**  If  *  stains,  but  are  decolorised  by  Gram's 

'-       V  method. 


Cultivation,  —  It  grows  readily  on 

FIG.   69. -Bacillus   pyocyaneus;    young     all    the  ordinary  media  at    the  room 
culture  on  agar.  .     .         ... 

Stained  with  weak  carbol-fuchsin.  x  1000.     temperature,  the  cultures  being  distin- 
guished by  the  formation  of  a  greenish 
pigment.     In  puncture  cultures  in  peptone-gelatin  a  greyish  line  appears  in 


MICROCOCCUS   TETRAGENUS.  1 87 

twenty-four  hours,  and  at  its  upper  part  a  small  cup  of  liquefaction  forms 
within  forty-eight  hours.  At  this  time  a  slightly  greenish  tint  is  seen  in  the 
superficial  part  of  the  gelatin.  The  liquefaction  extends  pretty  rapidly,  the 
fluid  portion  being  turbid  and  showing  masses  of  growth  at  its  lower  part. 
The  green  colour  becomes  more  and  more  marked  and  diffuses  through  the 
gelatin.  Ultimately  liquefaction  reaches  the  wall  of  the  tube.  In  plate  cul- 
tures the  colonies  appear  as  minute  whitish  points,  those  in  the  surface  being 
the  larger.  Under  a  low  power  of  the  microscope  they  have  a  brownish  yel- 
low colour  and  show  a  modulated  surface,  the  superficial  colonies  being  thinner 
and  larger.  Liquefaction  soon  occurs,  the  colonies  on  the  surface  forming 
shallow  cups  with  small  irregular  masses  of  growth  at  the  bottom,  the  deep 
colonies  small  spheres  of  liquefaction.  Around  the  colonies  a  greenish  tint 
appears.  On  agar  the  growth  forms  an  abundant  slimy  greyish  layer  which 
afterwards  becomes  greenish  and  has  a  metallic  sheen,  and  a  bright  green 
colour  diffuses  through  the  whole  substance  of  the  medium.  On  potatoes  the 
growth  is  an  abundant  reddish-brown  layer  resembling  that  of  the  glanders 
bacillus,  and  the  potato  sometimes  shows  a  greenish  discoloration.  Milk  is 
slightly  acidified,  and  peptonised  without  coagulation  as  a  rule. 

From  the  cultures  there  can  be  extracted  by  chloroform  a  coloured  body 
pyocyanin,  which  belongs  to  the  aromatic  series,  and  crystallises  in  the  form 
of  long,  delicate  bluish-green  needles.  On  the  addition  of  a  weak  acid  its 
colour  changes  to  a  redr 

In  man,  many  observers  have  described  it  as  being  associated  with 
abscess  formation,  pericarditis,  pyelitis,  dysentery,  etc.  It  has  likewise  dis- 
tinct pathogenic  action  in  certain  animals.  Subcutaneous  injection  of  small 
doses  in  rabbits  may  produce  a  local  suppuration,  but  if  the  dose  be  large, 
spreading  haemorrhagic  oedema  results,  which  may  be  attended  by  septicaemia. 

Intravenous  injection  may  pro- 
duce, according  to  the  dose,  rapid 
septicaemia  with  nephritis,  or  some- 
times a  more  chronic  condition  of  ^  «w  v 
wasting  attended  by  albuminuria.  ?  * 

Micrococcus     tetragenus.  —  This  & 

organism,  first  described  by  Gaffky,  %  t 

is  characterised    by  the  fact  that  it  Si  * 

divides  in  two  planes  at  right  angles          f* 
to  one  another  (Fig.  70),  and  is  thus  ., 

generally  found  in  the  tissues  in  groups  f 

of   four  or  tetrads,  which  are  often  m  f 

seen  to  be  surrounded  by  a  capsule.  +    fr         * 

The  cocci  measure  i  //,  in  diameter.  <l 

They  stain  readily  with  all  the  ordi- 
nary stains,  and  also  retain  the  stain          FIG.  70.  — Micrococcus  tetragenus;  young 
in  rViv«'o  * ,   *i     A  culture  on  agar,  showing  tetrads. 

m  Gram  s  method.  Stained  ^  weak  carbol.fuchsin.    x  Iocxx 

It  grows  readily  on  all  the  media 

at  the  room  temperature.  In  a  puncture  culture  on  peptone-gelatin  a  pretty 
thick  whitish  line  forms  along  the  track  of  the  needle,  whilst  on  the  surface 
there  is  a  thick  rounded  disc  of  whitish  colour.  The  gelatin  is  not  liquefied. 


1 88     INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 

On  the  surface  of  agar  and  of  potato  the  growth  is  an  abundant  moist  layer 
of  the  same  colour.  The  growth  on  all  the  media  has  a  peculiar  viscid  or 
tenacious  character,  owing  to  the  gelatinous  character  of  the  sheaths  of  the 
cocci. 

White  mice  are  exceedingly  susceptible  to  this  organism.  Subcutaneous 
injection  is  followed  by  a  general  septicaemia,  the  .organism  being  found  in 
large  numbers  in  the  blood  throughout  the  body.  Guinea-pigs  are  less  sus- 
ceptible ;  sometimes  only  a  local  abscess  with  a  good  deal  of  necrotic  change 
results  ;  sometimes  there  is  also  septicaemia. 

Diplococcus  intracellularis  meningitidis.  —  This  organism  was  first  found 
by  Weichselbaum  in  the  purulent  exudate  in  a  number  of  cases  of  cerebro- 
spinal  meningitis,  and  has  since  been  found  by  other  observers  in  some  epi- 
demics of  the  disease.  It  occurs  in  large  numbers  in  the  pus  in  the  form  of 
a  rounded  or  oval  diplococcus  (with  the  long  axis  lying  transversely),  chiefly 
in  the  interior  of  leucocytes  (Fig.  71).  In  fact,  it  closely  resembles  the  gono- 

coccus  both  in  morphological  charac- 
ters and  in  arrangement.  Like  the 
latter  also  it  loses  the  stain  in  Gram's 
method.  Its  conditions  of  growth 
outside  the  body  are  somewhat  limited. 
It  grows  best  on  glycerin  agar  and 
Lofner's  blood  serum,  forming  a  num- 
ber of  transparent  colonies  which  run 
together  to  form  a  thin  layer.  Growth 
occurs  most  rapidly  at  the  tempera- 
ture of  the  body,  and  entirely  ceases 
at  the  ordinary  room  temperature. 
Individual  cultures  die  out  after  two 
to  six  days,  but  growth  can  be  main- 
tained indefinitely  in  successive  sub- 

FIG.  71.  —  Film  preparation  of  exudation     cultures.       Inoculation     by    ordinary 

methods  shows  that  it  has  little  viru- 

Stained  with  carbol-thionin-blue.    xiooo.     lence    for    guinea-pigs,    rabbits,    etc. 

A  number  of  experiments  have  been 

performed  by  introducing  pure  cultures  under  the  dura,  and  in  some  cases 
meningitis  and  encephalitis  have  resulted,  but  the  disease  as  it  affects  the 
human  subject  is  not  fully  reproduced.  From  the  constancy  with  which  it 
has  been  found  in  the  various  cases  of  some  epidemics  there  can  be  little 
doubt  that  it  is  the  causal  agent  in  a  certain  proportion  of  cases  of  cerebro- 
spinal  meningitis  (p.  201).  It  is  of  interest  to  note  that  in  a  considerable 
number  of  such  cases  it  has  been  detected  during  the  disease  in  the  nasal 
secretion,  whereas  in  normal  individuals  it  is  very  rare. 

Bacillus  acnes.  —  According  to  Gilchrist  this  organism  is  the  undoubted 
cause  of  a  pustular  disease  of  the  skin  known  as  acne  vulgaris.  The  morphol- 
ogy of  the  organism  as  seen  in  smear  preparations  made  direct  from  the  pus 
of  an  acne  nodule,  resembles  very  closely  that  of  B.  coli,  the  bacilli  being 
short  and  plump  with  rounded  ends ;  very  rarely  branching  forms  are  met 
with,  although  in  old  cultures  such  forms  are  not  uncommon.  The  bacilli 


EXPERIMENTAL   INOCULATION.  189 

stain  readily  with  the  common  aniline  dyes,  occasionally  showing  plasmolytic 
vacuolation ;  they  retain  the  stain  in  Gram's  method.  They  are  non-motile, 
but  Brownian  movement  is  very  active.  No  spores  have  been  observed. 
Growth  occurs  best  at  37°  C.  on  glycerin  agar,  whose  reaction  is  3  •  o  4-  to  phe- 
nol-phthaleine,  on  blood  serum  (Lbffler's),  and  in  bouillon,  less  frequently  in 
litmus  milk.  No  growth  is  noticeable  on  potato,  gelatin,  or  in  Dunham's 
medium.  For  successful  isolation  from  an  acne  node,  glycerin  agar  or  Lof- 
flers  blood  serum  should  be  used,  and  the  material  from  the  nodule  should 
not  be  smeared  over  the  surface  but  deposited  in  pieces  about  2  c.mm.  and 
allowed  to  incubate  a  week  or  ten  days.  Then  these  small  whitish  masses 
(which  are  in  truth  the  bacilli  themselves)  will  be  found  to  have  increased  in 
bulk  and  thrown  out  an  extending  grey  zone  of  growth ;  their  consistency  is 
somewhat  pultaceous,  and  they  may  be  moved' about  en  masse  with  the  needle. 
Old  cultures  take  on  a  pink  or  light  maroon  colour,  or  sometimes  a  yellpw-drab. 

In  well-marked  cases  of  the  disease  the  blood  of  the  patient  will  cause 
agglutination  of  the  bacilli  in  dilutions  of  1-50,  1-60,  and  i-ioo. 

The  organism  is  pathogenic  to  mice  and  guinea-pigs,  either  upon  subcu- 
taneous or  intraperitoneal  inoculation. 

Experimental  Inoculation. — We  shall  consider  chiefly  the 
staphylococcus  pyogenes  aureus  and  the  streptococcus  pyogenes, 
as  these  have  been  most  fully  studied. 

It  may  be  stated  at  the  outset  that  the  occurrence  of  suppu- 
ration depends  upon  the  number  of  organisms  introduced  into 
the  tissues,  the  number  necessary  varying  not  only  in  different 
animals,  but  also  in  different  parts  of  the  same  animal,  a  smaller 
number  producing  suppuration  in  the  anterior  chamber  of  the 
eye,  for  example,  than  in  the  peritoneum.  The  virulence  of  the 
organism  also  may  vary,  and  corresponding  results  may  be  pro- 
duced. Especially  is  this  so  in  the  case  of  the  streptococcus 
pyogenes. 

The  staphylococcus  aureus,  when  injected  subcutaneous  ly  in 
suitable  numbers,  produces  an  acute  local  inflammation  which  is 
followed  by  suppuration,  in  the  manner  described  above.  The 
spread  of  the  suppuration  goes  pari  passit  with  the  growth  of 
the  cocci.  Wherever  the  condition  is  spreading  the  cocci  are 
present  in  the  tissues  at  the  margin,  but  after  it  has  ceased  to 
spread  they  are  practically  confined  to  the  pus.  In  the  latter 
case  reaction  occurs  on  the  part  of  the  connective  tissues  in  the 
form  of  cellular  proliferation  and  formation  of  new  capillaries, 
which  lead  to  the  formation  of  a  granulation  tissue  barrier.  If 
a  large  dose  is  injected,  the  cocci  may  enter  the  blood  stream  in 
sufficient  numbers  to  cause  secondary  suppurative  foci  in  inter- 
nal organs  (cf.  intravenous  injection). 


IQO    INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 

Intravenous  injection  in  rabbits,  for  example,  produces  inter- 
esting results  which  vary  according  to  the  quantity  used.  If  a  con- 
siderable quantity  be  injected,  the  animal  may  die  in  twenty-four 
hours  of  a  general  septicaemia,  numerous  cocci  being  present  in 
the  capillaries  of  the  various  organs,  often  forming  plugs.  If  a 
smaller  quantity  be  used,  the  cocci  gradually  disappear  from  the 
circulating  blood  ;  some  become  destroyed,  while  others  settle  in 
the  capillary  walls  in  various  parts  and  produce  minute  abscesses. 
These  are  most  common  in  the  kidneys,  where  they  occur  both 
in  the  cortex  and  medulla  as  minute  yellowish  areas  surrounded 
by  a  zone  of  intense  congestion  and  haemorrhage.  Similar  small 
abscesses  may  be  produced  in  the  heart  wall,  in  the  liver,  under 
the  periosteum,  and  in  the  interior  of  bones,  and  occasionally  in 
the  striped  muscles.  Very  rarely  indeed,  in  experimental  injec- 
tion, do  the  cocci  settle  on  the  healthy  valves  of  the  heart.  If, 
however,  when  the  organisms  are  injected  into  the  blood,  there 
be  any  traumatism  of  a  valve,  or  of  any  other  part  of  the  body, 
they  show  a  special  tendency  to  settle  at  these  weakened  points. 

Experiments  on  the  hitman  subject  have  also  proved  the  pyo- 
genic  properties  of  those  organisms.  Garre  inoculated  scratches 
near  the  root  of  his  finger-nail  with  a  pure  culture,  a  small  cuta- 
neous pustule  resulting ;  and  by  rubbing  a  culture  over  the  skin 
of  the  forearm  he  caused  a  carbuncular  condition  which  healed 
only  after  some  weeks.  Confirmatory  experiments  of  this  nature 
have  been  made  by  Bockhart,  Bumm,  and  others.  Harris  has 
observed  an  infection  to  occur  upon  the  palmar  surfaces  of  the 
fingers  of  both  hands,  the  organisms  having,  in  the  absence  of 
any  demonstrable  lesion,  passed  down  the  sweat  ducts  to  the 
deeper  lying  tissues. 

When  tested  experimentally  the  staphylococcus  pyogenes 
albus  has  practically  the  same  pathogenic  effects  as  the  staphy- 
lococcus aureus. 

The  streptococcus  pyogenes  is  an  organism  the  virulence  of 
which  varies  much  according  to  the  diseased  condition  from 
which  it  has  been  obtained,  and  also  one  which  loses  its  viru- 
lence rapidly  in  cultures.  Even  highly  virulent  cultures,  if  grown 
under  ordinary  conditions,  in  the  course  of  time  lose  practically 
all  pathogenic  power.  By  passage  from  animal  to  animal,  how- 
ever, the  virulence  may  be  much  increased,  and  pari  passu  the 
effects  of  inoculation  are  correspondingly  varied.  Marmorek, 


VARIETIES   OF    STREPTOCOCCI. 


IQI 


for  example,  found  that  the  virulence  of  a  streptococcus  can  be 
enormously  increased  by  growing  it  alternately  (a]  in  a  mixture 
of  human  blood  serum  and  bouillon  (vide  p.  46),  and  (b)  in 
the  body  of  a  rabbit ;  ultimately,  after  several  passages  it  pos- 
sesses a  super-virulent  character,  so  that  even  an  extremely  minute 
dose  introduced  into  the  tissues  of  a  rabbit  produces  rapid  septi- 
caemia, with  death  in  a  few  hours.  It  has  been  proved  by  Mar- 
morek's  experiments,  and  those  of  others,  that  the  same  species 
of  streptococcus  may  produce  at  one  time  merely  a  passing  local 
redness,  at  another  a  local  suppuration,  at  another  a  spreading 
erysipelatous  condition,  or  again  a  general  septicaemic  infection, 
according  as  its  virulence  is  artificially  increased.  Such  experi- 
ments are  of  extreme  importance  as  explaining  to  some  extent 
the  great  diversity  of  lesions  in  the  human  subject  with  which 
streptococci  are  associated. 

Varieties  of  Streptococci.  —  Formerly  the  streptococcus  pyo- 
genes  and  the  streptococcus  erysipelatis  were  regarded  as  two 
distinct  species,  and  various  points  of  difference  between  them 
were  given.  Further  study,  and  especially  the  results  obtained 
by  modifying  the  virulence,  have  shown  that  these  distinctions 
cannot  be  maintained,  and  now  nearly  all  authorities  are  agreed 
that  the  two  organisms  are  one  and  the  same,  erysipelas  being 
produced  when  the  streptococcus  pyogenes  of  a  certain  standard 
of  virulence  gains  entrance  to  the  lymphatics  of  the  skin. 
Petruschky,  moreover,  has  shown  conclusively  that  a  strepto- 
coccus cultivated  from  pus  may  cause  erysipelas  in  the  human 
subject.  He  obtained  a  pure  culture  of  a  streptococcus  from  a 
case  of  purulent  peritonitis  secondary  to  parametritis,  the  patient 
having  never  suffered  from  erysipelas.  By  inoculations  with 
this  culture  he  produced  typical  erysipelas  in  two  women  suf- 
fering from  cancer. 

More  recently  a  distinction  has  been  drawn  between  a  streptococcus  longus, 
which  forms  long  chains,  and  is  pathogenic  to  rabbits  or  mice,  and  a  strepto- 
coccus brevis,  which  occurs  in  the  mouth  in  normal  conditions,  and  is  without 
pathogenic  properties  when  tested  experimentally.  The  growth  of  the  former 
in  bouillon  forms  a  somewhat  granular  deposit,  that  of  the  latter  a  more 
abundant  and  flocculent  deposit.  Marmorek  has,  however,  found  that  the 
same  streptococcus  may  at  one  time  grow  in  short,  at  another  in  long  chains, 
and  Kolle  has  shown  that  a  streptococcus,  which  originally  grew  in  long 
chains,  formed  only  short  chains  after  being  repeatedly  passed  through  the 
body  of  the  mouse,  the  appearance  of  the  growth  in  bouillon  being  corre- 


192     INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 

spondingly  altered  (p.  186).  There  does  not,  therefore,  seem  at  present  suffi- 
cient evidence  for  looking  upon  these  two  varieties  as  distinct  species.  It  is 
sufficient  to  bear  in  mind  that  streptococci  in  the  normal  mouth  are  usually  non- 
virulent,  and  grow  in  short  chains.  On  the  other  hand,  in  some  cases  of  very 
virulent  streptococcus  infection  in  the  human  subject  we  have  found  the  organ- 
ism occurring  only  in  very  short  chains.  The  streptococcus  conglomerate,  so 
called  from  the  appearance  of  the  growth  in  bouillon,  is  to  be  regarded  merely 
as  another  variety,  which  forms  very  long  chains  and  is  usually  possessed  of  a 
high  degree  of  virul'ence,  though  its  distinctive  characters  are  not  permanent. 
It  has  often  been  obtained  from  the  fauces  in  scarlet  fever. 

We  may  accordingly  conclude  that,  though  it  cannot  be 
definitely  stated  that  all  the  streptococci  concerned  in  the  pro- 
duction of  disease  in  the  human  subject  are  of  the  same  species, 
we  have  not  the  means  of  classifying  them  as  distinct  species. 

Bacillus  coli  communis.  —  The  virulence  of  this  organism 
also  varies  much  and  can  be  increased  by  passage  from  animal 
to  animal.  Injection  into  the  serous  cavities  of  rabbits  pro- 
duces a  fibrinous  inflammation  which  becomes  purulent  if  the 
animal  lives  sufficiently  long.  If,  however,  the  virulence  of  the 
organism  be  of  a  high  order,  death  takes  place  before  suppura- 
tion is  established,  and  there  is  a  septicaemic  condition,  the 
organisms  occurring  in  large  numbers  in  the  blood.  Intra- 
venous injection  of  a  few  drops  of  a  virulent  bouillon  culture 
usually  produces  a  rapid  septicaemia  with  scattered  haemorrhages 
in  various  organs. 

Other  Effects.  —  It  has  been  found  by  independent  observers  that  in  cases 
where  rabbits  recover  after  intravenous  injection  of  bacillus  coli  communis,  a 
certain  proportion  suffer  from  paralysis  and  sometimes  from  atrophy  of  mus- 
cles, especially  of  the  posterior  limbs,  these  symptoms  being  due  to  lesions  of 
the  cells  in  the  anterior  cornua  of  the  spinal  cord.  Somewhat  similar  results 
have  been  obtained  by  others  after  inoculations  with  staphylococci  and  strepto- 
cocci, a  certain  proportion  only  of  the  animals  showing  paralytic  symptoms 
and  corresponding  changes  in  the  spinal  cord.  The  lesions  are  believed  to 
be  due  chiefly  to  the  action  of  the  products  of  the  organisms  on  the  highly 
organised  nervous  elements.  Much  further  research  requires  to  be  done 
before  the  importance  of  these  results  can  be  properly  estimated,  but  it  is  not 
improbable  that  they  will  throw  light  on  the  causation  of  nervous  lesions  which 
occur  in  the  human  subject,  and  the  etiology  of  which  at  present  is  quite 
obscure.  Some  observers,  chiefly  of  the  French  school,  consider  that  paralysis 
associated  with  cystitis,  in  which  the  bacillus  coli  communis  is  often  present, 
may  have  such  a  causation,  and  that  paralytic  conditions  following  acute 
infective  fevers  may  be  produced  by  the  products  of  pyogenic  cocci,  which 
frequently  occur  in  these  conditions. 


LESIONS    IN   THE   HUMAN    SUBJECT.  193 

Lesions  in  the  Human  Subject.  —  The  following  statement 
may  be  made  with  regard  to  the  occurrence  of  the  chief  organ- 
isms mentioned,  in  the  various  suppurative  and  inflammatory 
conditions  in  the  human  subject.  The  account  is,  however,  by 
no  means  exhaustive,  as  clinical  bacteriology  has  shown  that 
practically  every  part  of  the  body  may  be  the  site  of  a  lesion 
produced  by  the  pyogenic  bacteria.  It  may  also  be  noted  that 
acute  catarrhal  conditions  of  cavities  or  tubes  are  in  many  cases 
also  to  be  ascribed  to  their  presence. 

The  staphylococci  are  the  most  common  causal  agents  in 
localised  abscesses,  in  pustules  on  the  skin,  in  carbuncles,  boils, 
etc.,  in  acute  suppurative  periostitis,  in  catarrhs  of  mucous  sur- 
faces, in  ulcerative  endocarditis,  and  in  various  pyaemic  condi- 
tions. They  may  also  be  present  in  septicaemia. 

Streptococci  are  especially  found  in  spreading  inflammation 
with  or  without  suppuration ;  in  diffuse  phlegmonous  and  ery- 
sipelatous  conditions,  suppurations  in  serous  membranes  and  in 
joints  (Fig.  72).  They  also  occur  in  acute  suppurative  periostitis 
and  ulcerative  endocarditis. 
Secondary  abscesses  in  lym- 
phatic glands  and  lymphangitis 
are  also,  we  believe,  more  fre- 
quently caused  by  streptococci 
than  staphylococci.  They  also 
produce  fibrinous  exudation  on 
the  mucous  surfaces,  leading 
to  the  formation  of  false  mem- 
brane in  many  of  the  cases  of 
non-diphtheritic  inflammation 
of  the  throat,  which  are  met 
with  in  scarlatina1  and  other  FIG.  72.  — streptococci  in  acute  suppu- 

COllditions,    and    they    are    also      ration-    Corrosive  film;  stained  by  Gram's 

method  and  safranin.     X  1000. 

the  organisms  most  frequently 

present  in  acute  catarrhal  inflammations  of  this  situation.  In 
puerperal  peritonitis  they  are  frequently  found  in  a  condition  of 
purity,  and  they  also  appear  to  be  the  most  frequent  cause  of 
puerperal  septicaemia,  in  which  condition  they  may  be  found 
after  death  in  the  capillaries  of  various  organs.  In  pyaemia 

1  True  diphtheria  may  also  occasionally  be  associated  with  this  disease,  usually 
as  a  sequel, 
o 


194     INFLAMMATORY   AND    SUFPURAT1VE   CONDITIONS. 

they  are  frequently  present,  though  in  most  cases  associated 
with  other  pyogenic  organisms.  Some  cases  of  enteritis  in 
infants  —  streptococcic  enteritis  —  are  also  apparently  due  to 
a  streptococcus,  which,  however,  presents  in  cultures  certain 
points  of  difference  from  the  streptococcus  pyogenes. 

The  bacillus  coli  communis  is  found  in  a  great  many  inflam- 
matory and  suppurative  conditions  in  connection  with  the 
alimentary  tract  —  for  example,  in  suppuration  in  the  perito- 
neum, or  in  the  extraperitoneal  tissue  with  or  without  perforation 
of  the  bowel,  in  the  peritonitis  following  strangulation  of  the 
bowel,  in  appendicitis  and  the  lesions  following  it,  in  suppura- 
tion around  the  bile-ducts,  etc.  It  may  also  occur  in  lesions  in 
other  parts  of  the  body, — endocarditis,  pleurisy,  etc.,  which  in 
some  cases  are  associated  with  lesions  of  the  intestine,  though 
in  others  such  cannot  be  found.  It  is  also  frequently  present  in 
inflammation  of  the  urinary  passages,  cystitis,  pyelitis,  abscesses 
in  the  kidneys,  etc.,  these  lesions  being  in  fact  most  frequently 
caused  by  this  or  closely  allied  organisms. 

In  certain  cases  of  enteritis  it  is  probably  the  causal  agent, 
though  this  is  difficult  of  proof,  as  it  is  much  increased  in 
numbers  in  practically  all  abnormal  conditions  of  the  intestine. 
We  may  remark  that  it  has  been  repeatedly  proved  that  the 
bacillus  coli  cultivated  from  various  lesions  is  more  virulent  than 
that  in  the  intestine,  its  virulence  having  been  heightened  by 
growth  in  the  tissues. 

The  micrococcus  tetragenus  is  often  found  in  suppurations 
in  the  region  of  the  mouth  or  in  the  neck,  and  also  occurs  in 
various  lesions  of  the  respiratory  tract,  in  phthisical  cavities, 
abscesses  in  the  lungs,  etc.  Sometimes  it  is  present  alone,  and 
probably  has  a  pyogenic  action  in  the  human  subject  under 
certain  conditions.  In  other  cases  it  is  associated  with  other 
organisms.  Recently  one  or  two  cases  of  pyaemia  have  been 
described  in  which  this  organism  was  found  in  a  state  of  purity 
in  the  pus  in  various  situations.  In  this  latter  condition  the  pus 
has  been  described  as  possessing  an  oily,  viscous  character,  and 
as  being  often  blood-stained. 

The  bacillus  pyocyaneus  is  rarely  found  alone  in  pus,  though 
it  is  not  infrequent  along  with  other  organisms.  We  have  met 
with  it  twice  in  cases  of  multiple  abscesses,  in  association  with 
the  staphylococcus  pyogenes  aureus.  Lately  some  diseases  in 


ENTRANCE   AND   SPREAD   OF   BACTERIA. 


195 


children  have  been  described  in  which  the  bacillus  pyocyaneus 
has  been  found  throughout  the  body  ;  in  these  cases  the  chief 
symptoms  have  been  fever,  gastro-intestinal  irritation,  pustular 
or  petechial  eruptions  on  the  skin,  and  general  marasmus. 

Suppurative  and  inflammatory  conditions,  associated  with 
the  organisms  of  special  diseases,  will  be  described  in  the  respec- 
tive chapters. 

Mode  of  Entrance  and  Spread.  —  Many  of  the  organisms  of 
suppuration  have  a  wide  distribution  in  nature,  and  many  also 

are    present   on 

***/*"•- 
the  skin  and  mu- 
cous membranes 

of  healthy  indi-  .      •: '.'/. 

viduals.  Staphy- 
lococci  are  com- 
monly present  *  •,  '..'*"<-. 
on  the  skin,  and  -v^; 
also  occur  in  the 
throat  and  other  *.  ^  , 
parts,  and  strep-  .^ 
tococci  can  often 
be  cultivated 
from  the  secre- 
tions of  the  •'"  ' '  -  •'  : 

/.  i*+  ~      ••••.-     •      '•  .  ' 

mouth  in  normal  'Ufc*^*  •'•:  "" 

conditions.    The 

pneumococcus 

of  Fraenkel  and  ^IG'  ?3- —  Minute    focus    of    commencing    suppuration    in 

brain  —  case  of  acute  ulcerative  'endocarditis.     In   the  centre  a 

the      pneumoba-     small  haemorrhage;  to  right  side  dark  masses  of  staphylococci ; 

cillllS    Of     Fried-     Z0ne  of  leucocytes  at  periphery. 

Alum  carmine  and  Gram's  method.     X  50. 

lander  have  also 

been  found  in  the  mouth  and  in  the  nasal  cavity,  whilst  the 
bacillus  coli  communis  is  a  normal  inhabitant  of  the  intestinal 
tract.  The  entrance  of  these  organisms  into  the  deeper  tissues 
when  a  surface  lesion  occurs  can  be  readily  understood.  Their 
action  will,  of  course,  be  favoured  by  any  depressed  condition 
of  vitality.  Though  in  normal  conditions  the  blood  is  bac- 
terium-free, we  must  suppose  that  from  time  to  time  a  certain 
number  of  such  organisms  gain  entrance  to  it  from  trifling 
lesions  of  the  skin  or  mucous  surfaces,  the  possibilities  of 


ig6    INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 


•entrance  from  the  latter  being  especially  numerous.  In  most 
cases  they  are  killed  by  the  action  of  the  healthy  serum  or 
cells  of  the  body,  and  no  harm  results.  If,  however,  there  be 
a  local  weakness,  they  .may  settle  in  that  part  and  produce  sup- 
puration, and  from  this  other  parts  of  the  body  may  be  infected. 
Such  a  supposition  as  this  is  necessary  to  explain  many  inflam- 
matory and  suppurative  conditions  met  with  clinically.  In  some 
cases  of  multiple  suppurations  due  to  staphylococcus  infection, 
-which  we  have  had  the  opportunity  to  examine,  only  an  ap- 
_^^^^^__  parently  unim- 

portant surface 
lesion  was  pres- 
ent ;  whilst  in 
others  no  lesion 
could  be  found 
to  explain  the 
origin  of  the 
infection.  The 
tonsils  may  at 
certain  times  be 
the  portals  of 
entry  for  sundry 
bacteria  giving 
rise  to  suppura- 
tive conditions, 
or  to  those  of 
general  infec- 

FlG.  74. —  Secondary  infection  of  a  glomenilus  of  kidney  by  Tl^ 

the  staphylococcus  aureus,  in  a  case  of  ulcerative  endocarditis.  "Oil. 

The  cocci  (stained  darkly)  are  seen  plugging  the  capillaries  and  cryptO^cnctlC  has 
also  lying  free.     The  glomerulus  is  much  swollen,  infiltrated  by  v    j    i_ 

leucocytes,  and  partly  necrosed.  been  applied    by 

Paraffin  section ;  stained  by  Gram's  method  and  Bismarck-  some  writers  to 
brown.  X  300. 

such     cases     in 

which  the  original  point  of  infection  cannot  be  found,  but  its  use 
is  scarcely  necessary. 

The  paths  of  secondary  infection  may  be  conveniently  sum- 
marised thus  :  First,  by  lymphatics.  In  this  way  the  lymphatic 
glands  may  be  infected,  and  also  serous  sacs  in  relation  to  the 
organs  where  the  primary  lesion  exists.  Second,  by  natural 
channels,  such  as  the  ureters  and  the  bile-ducts,  the  spread 
being  generally  associated  with  an  inflammatory  condition  of  the 


••••' 


ULCERATIVE   ENDOCARDITIS.  197 

lining  epithelium.  In  this  way  the  kidneys  and  liver  respectively 
may  be  infected.  Third,  by  the  blood-vessels :  (a)  by  a  few 
organisms  gaining  entrance  to  the  blood  from  a  local  lesion,  and 
settling  in  a  favourable  nidus  or  a  damaged  tissue,  the  original 
path  of  infection  often  being  obscure ;  (£)  by  a  septic  phlebitis 
with  suppurative  softening  of.  the  thrombus  and  resulting  em- 
bolism ;  and  we  may  add  (c)  by  a  direct  extension  along  a  vein, 
producing  a  spreading  thrombosis  and  suppuration  within  the 
vein.  In  this  way  suppuration  may  spread  along  the  portal  vein 
to  the  liver  from  a  lesion  in  the  alimentary  canal,  the  condition 
being  known  as  pyelo-phlebitis  suppurativa. 

Although  many  of  the  lesions  produced  by  the  bacteria  under 
consideration  have  already  been  mentioned,  certain  conditions 
may  be  selected  for  further  consideration  on  account  of  their 
clinical  importance  or  bacteriological  interest. 

Ulcerative  Endocarditis.  — This  condition  has  been  proved  to 
be  a  bacterial  infection  of  the  valves  of  the  heart,  and  may  be 
produced  by  various  organisms,  chiefly  pyogenic.  Of  these  the 
staphylococci  and  streptococci  are  most  frequently  found.  In 
some  cases  of  ulcerative  endocarditis  following  pneumonia,  the 
pneumococcus  (Fraenkel's)  is  present ;  in  others  pyogenic  cocci, 
especially  streptococci.  Other  organisms  have  been  cultivated 
from  different  cases  of  the  disease,  and  some  of  these  have 
received  special  names ;  for  example,  the  diplococcus  endocardi- 
tidis  encapsulatus,  bacillus  endocarditidis  griseus  (Weichsel- 
baum),  micrococcus  zymogenes  (MacCallum  and  Hastings),  and 
others.  In  some  cases  the  bacillus  coli  communis  has  been 
found,  and  occasionally  in  endocarditis  following  typhoid  the 
typhoid  bacillus  has  been  described  as  the  organism  present, 
but  further  observations  on  this  point  are  desirable.  The  gono- 
coccus  also  has  been  shown  to  affect  the  heart  valves  (p.  230), 
though  this  is  a  very  rare  occurrence.  Tubercle  nodules  on  the 
heart  valves  have  been  found  in  a  few  cases  of  acute  tuber- 
culosis, though  no  vegetative  or  ulcerative  condition  is  produced. 

In  some  cases,  though  we  believe  not  often,  the  organisms 
may  attack  healthy  valves,  producing  a  primary  ulcerative  endo- 
carditis, but  more  frequently  the  valves  have  been  the  seat  of 
previous  endocarditis,  secondary  ulcerative  endocarditis  being 
thus  produced.  In  both  conditions  the  affection  of  the  valves 
usually  occurs  in  the  course  of  suppurative  or  inflammatory 


198     INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 


conditions  elsewhere,  e.g.  in  osteomyelitis,  in  septic  inflamma- 
tions of  the  urinary  passages,  in  pyaemia  and  septicaemia,  in  the 
course  of  or  following.  infective  fevers,  and  not  very  infrequently 
as  a  sequel  to  acute  pneumonia.  In  some  cases,  especially 
when  the  valves  have  been  previously  diseased,  the  source  of 
the  infection  is  quite  obscure.  It  is  evident  that  as  the  vege- 
tations are  composed  for  the  most  part  of  unorganised  mate 
rial,  they  do  not  offer  the  same  resistance  to  the  growth  of 

bacteria,  when 

j£  ;  a     few     reach 

them,  as  a 
healthy  cellu- 
lar tissue  does. 
On  micro- 
scopic exami- 
nation of  the 
diseased  valves 
the  organisms 
are  usually  to 
be  found  in 
enormous  num- 
bers, some- 
times forming 
an  almost  con- 
tinuous layer 
on  the  surface, 

or  OCCUlTing  in 

maSSCS 
clusters     in 

spaces    in    the 

vegetation  (Fig.  75).  By  their  action  a  certain  amount  of 
softening  or  breaking  down  of  the  vegetations  occurs,  and  the 
emboli  thus  produced  act  as  the  carriers  of  infection  to  other 
organs,  and  give  rise  to  secondary  suppurations. 

Experimental.  —  Occasionally  ulcerative  endocarditis  is  produced  by  the 
simple  intravenous  injection  of  staphylococci  and  streptococci  into  the  circula- 
tion, but  this  is  a  very  rare  occurrence.  It  often  follows,  however,  when  the 
valves  have  been  previously  injured.  Orth  and  Wyssokowitsch  at  a  com- 
paratively early  date  produced  the  condition  by  damaging  the  aortic  cusps  by 
a  glass  rod  introduced  through  the  carotid,  and  afterwards  injecting  staphylo- 


FIG.  75.  —  Section  of  a  vegetation  in  ulcerative  endocarditis, 
showing  numerous  staphylococci  lying  in  the  spaces.  The  lower 
portion  is  a  fragment  in  process  of  separation. 

Stained  by  Gram's  method  and  Bismarck-brown.     X  600. 


PERIOSTITIS,  OSTEOMYELITIS,  ERYSIPELAS.  199 

cocci  into  the  circulation.  Similar  experiments  have  since  been  repeated 
with  streptococci,  pneumococci,  and  other  organisms,  with  like  result.  Ribbert 
found  that  if  a  potato  culture  of  the  staphylococcus  aureus  were  rubbed  down 
so  as  to  form  an  emulsion  in  salt  solution,  and  then  injected  into  the  circula- 
tion, some  minute  fragments  became  arrested  at  the  attachment  of  the  chordae 
tendineae  and  produced  an  ulcerative  endocarditis. 

Acute  Suppurative  Periostitis  and  Osteomyelitis.  —  Special 
mention  is  made  of  this  condition  on  account  of  its  comparative 
frequency  and  gravity.  The  great  majority  of  cases  are  caused 
by  the  pyogenic  cocci,  of  which  one  or  two  varieties  may  be 
present,  the  staphylococcus  aureus,  however,  occurring  most 
frequently.  Pneumococci  have  been  found  alone  in  some  cases, 
and  in  a  few  cases  following  typhoid  fever,  apparently  well 
authenticated,  the  typhoid  bacillus  has  been  found  alone.  In 
others  again  the  bacillus  coli  communis  is  present. 

The  affection  of  the  periosteum  or  interior  of  the  bones  by 
these  organisms,  which  is  especially  common  in  young  subjects, 
may  take  place  in  the  course  of  other  affections  produced  by 
these  organisms  or  in  the  course  of  infective  fevers,  but  in  a 
gr~at  many  cases  the  path  of  entrance  cannot  be  determined. 
In  the  course  of  this  disease  serious  secondary  infections  are 
always  very  liable  to  follow,  such  as  small  abscesses  in  the 
kidneys,  heart  wall,  lungs,  liver,  etc.,  suppurations  in  serous 
cavities,  and  ulcerative  endocarditis  ;  in  fact,  some  cases  present 
the  most  typical  examples  of  extreme  general  staphylococcus 
infection.  The  entrance  of  the  organisms  into  the  blood  stream 
from  the  lesion  of  the  bone  is  especially  favoured  by  the  arrange- 
ment of  the  veins  in  the  bone  and  marrow. 

Experimental.  —  Multiple  abscesses  in  the  bones  and  under  the  periosteum 
may  occur  in  simple  intravenous  injection  of  the  pyogenic  cocci  into  the 
blood,  and  are  especially  liable  to  be  formed  when  young  animals  are  used. 
These  abscesses  are  of  small  size,  and  do  not  spread  in  the  same  way  as  in 
the  natural  disease  in  the  human  subject. 

In  experiments  on  healthy  animals,  however,  the  conditions  are  not 
analogous  to  those  of  the  natural  disease.  We  must  presume  that  in  the 
latter  there  is  some  local  weakness  or  susceptibility  which  enables  the  few 
organisms  which  have  reached  the  part  by  the  blood  to  settle  and  multiply. 
Moreover,  if  a  bone  be  experimentally  injured,  e.g.  by  actual  fracture  or  by 
stripping  off  the  periosteum,  before  the  organisms  are  injected,  then  a  much 
more  extensive  suppuration  occurs  at  the  injured  part. 

Erysipelas.  —  A  spreading  inflammatory  condition  of  the 
skin  may  be  produced  by  a  variety  of  organisms,  but  the  disease 


200    INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 

in  the  human  subject  in  its  characteristic  form  is  almost  invari- 
ably due  to  a  streptococcus,  as  was  shown  by  Fehleisen  in  1884. 
He  obtained  pure  cultures  of  the  organism,  and  gave  it  the 
name  of  streptococcus  erysipelatis  ;  and,  further,  by  inoculations 
on  the  human  subject  as  a  therapeutic  measure  in  malignant 
disease,  he  was  able  to  reproduce  erysipelas.  As  stated  above, 
however,  one  after  another  of  the  supposed  points  of  difference 
between  the  streptococcus  of  erysipelas  and  that  of  suppuration 
has  broken  down,  and  it  is  now  generally  held  that  erysipelas  is 
produced  by  the  streptococcus  pyogenes  of  a  certain  degree  of 
virulence.  It  must  be  noted,  however,  that  erysipelas  passes 
from  patient  to  patient  as  erysipelas,  and  purulent  conditions 
due  to  streptococci  do  not  appear  liable  to  be  followed  by  ery- 
sipelas. On  the  other  hand,  the  connection  between  erysipelas 
and  puerperal  septicaemia  is  well  established  clinically. 

In  a  case  of  erysipelas  the  streptococci  are  found  in  large 
numbers  in  the  lymphatics  of  the  cutis  and  underlying  tissues, 
just  beyond  the  swollen  margin  of  the  inflammatory  area.  As 
the  inflammation  advances  they  gradually  die  out,  and  after  a 
time  their  extension  at  the  periphery  comes  to  an  end.  The 
streptococci  may  extend  to  serous  and  synovial  cavities  and  set 
up  inflammatory  or  suppurative  change,  —  peritonitis,  meningitis, 
and  synovitis  may  thus  be  produced. 

Meningitis. — Although  almost  any  of  the  above-mentioned 
pyogenic  organisms  may  be  concerned  in  the  causation  of 
meningitis,  some  general  statements  may  be  made  regarding  it. 
A  considerable  number  of  cases  of  meningitis,  especially  in  chil- 
dren, are  due  to  the  pneumococcus.  In  many  instances  where 
no  other  lesions  are  present  the  extension  is  by  the  Eustachian 
tube  to  the  middle  ear  and  thence  to  the  brain ;  in  other  cases 
the  path  of  infection  is  by  means  of  the  blood  stream,  usually 
from  some  inflammatory  lesion  in  the  lungs.  Meningitis  is  not 
infrequently  produced  by  a  streptococcus,  especially  when  middle 
ear  disease  is  present,  less  frequently  by  one  of  the  staphylococci. 
Occasionally  more  than  one  organism  may  be  concerned.  In 
meningitis  following  influenza,  the  influenza  bacillus  has  been 
found  in  a  few  cases,  and  in  tubercular  meningitis  the  tubercle 
bacillus  of  course  is  present,  especially  in  the  nodules  along  the 
sheaths  of  the  vessels.  The  pneumobacillus  and  B.  typhosus 
also  have  been  found  in  a  few  cases  of  meningitis. 


CONJUNCTIVITIS.  2OI 

In  a  number  of  the  earlier  cases  of  epidemic  cerebro-spinal 
meningitis  the  pneumococcus  was  described  as  the  organism 
present,  but  later  observations  made  in  different  parts  of  the 
world  show  that  the  organism  usually  concerned  is  undoubtedly 
the  diplococcus  intracellularis  meningitidis.  In  acute  cases,  and 
especially  in  the  earlier  stages,  it  is  usually  present  in  large 
numbers,  but  in  the  more  chronic  it  occurs  sparsely,  and  its 
presence  may  be  demonstrated  only  with  difficulty.  The  organ- 
ism can  usually  be  obtained  by  means  of  lumbar  puncture.  Cer- 
tain sporadic  cases  of  meningitis  are  also  due  to  this  organism, 
and  it  is  extremely  probable  that  the  diplococcus  of  simple  basal 
meningitis  in  children  described  by  Still  is  merely  a  modification 
of  this  meningococcus. 

Conjunctivitis.  —  A  considerable  number  of  organisms  are 
concerned  in  the  production  of  conjunctivitis  and  its  associated 
lesions.  Of  these  a  number  appear  to  be  specially  associated 
with  this  region.  Thus  a  small  organism,  generally  known  as 
the  Koch-Weeks  bacillus,  is  the  most  common  cause  of  acute 
contagious  conjunctivitis,  especially  prevalent  in  Egypt,  but  also 
having  a  very  wide  distribution.  This  organism  morphologically 
resembles  the  influenza  bacillus,  and  its  conditions  of  growth 
are  even  more  restricted,  as  it  rarely  grows  on  blood  agar,  the 
best  medium  being  serum  agar.  Another  organism  exceedingly 
like  the  previous,  apparently  differing  from  it  only  in  the  rather 
wider  conditions  of  growth,  is  Muller's  bacillus.  It  has  been  culti- 
vated by  him  in  a  considerable  proportion  of  cases  of  trachoma, 
but  its  relation  to  this  condition  is  still  a  matter  of  dispute. 
Another  bacillus  which  is  now  well  recognised  is  the  diplo-bacil- 
lus  of  conjunctivitis  first  described  by  Morax.  It  is  especially 
common  in  the  more  subacute  cases  of  conjunctivitis.  Eyre 
found  it  in  2.5  per  cent  of  all  cases  of  conjunctivitis.  Its  cultural 
characters  are  given  below.  The  xerosis  bacillus  (Chap.  XVI.) 
has  been  found  in  xerosis  of  the  conjunctiva,  in  follicular  con- 
junctivitis, and  in  other  conditions ;  it  appears  to  occur  some- 
times also  in  the  normal  conjunctiva.  Acute  conjunctivitis  is 
also  produced  by  the  pneumococcus,  epidemics  of  the  disease 
being  sometimes  due  to  this  organism,  and  also  by  streptococci 
and  staphylococci.  True  diphtheria  of  the  conjunctiva  caused 
by  the  Klebs-Lbfrler  bacillus  also  occurs,  whilst  in  gonorrhoeal 
conjunctivitis,  often  of  an  acute  purulent  type,  the  gonococcus 
is  present. 


202     INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 


Diplo-bacillus  of  Conjunctivitis.  —  This  organism,  discovered  by  Morax, 
is  a  small  plump  bacillus,  measuring  I  x  2  /x,  and  usually  occurring  in  pairs 

(Fig.  76).  It  is  non-motile,  does 
not  form  spores,  and  is  decolor- 
ised by  Gram's  method.  .  It  does 
not  grow  on  the  ordinary  gelatin 
and  agar  media,  the  addition  of 
blood  or  serum  being  necessary. 
On  serum  it  forms  small  rounded 
colonies  which  produce  small  pits 
of  liquefaction  ;  hence  it  is  some- 
times called  the  bacillus  lacuna- 
tus.  In  cultures  it  is  distinctly 
pleomorphous,  and  involution 
forms  also  occur.  It  it  non-patho- 
genic to  the  lower  animals. 

Acute    Rheumatism.  — 

There  are  many  facts  which 

FIG.  76.  — Film  preparation  of  conjunctiva!  seem  to  indicate  the  infec- 
dipl°-bacillus  °f  conjunc>  tive  nature  of  this  disease, 

and     investigations     from 

this  point  of  view  have  yielded  results  of  which  mention  may 
here  be  made.  A  number  of  organisms  have  been  cultivated 
from  the  affected  tissues  by  different  observers,  and  have  been 
supposed  to  have  a  special  relation  to  the  disease. 

Achalme,  Thiroloix,  Bettencourt,  and  others  of  the  French 
school  describe  the  occurrence  of  an  anaerobic  bacillus,  similar 
in  appearance  to  B.  anthracis,  in  many  cases  of  acute  rheuma- 
tism, which  they  claim  bears  an  etiological  relationship  to  the 
disease.  Hewlett,  in  England,  in  the  only  case  he  examined, 
isolated  a  bacillus  similar  in  characteristics  to  that  of  Achalme. 
And  in  America,  Gwyn,  from  a  case  of  chorea  rheumatica, 
isolated  an  anaerobic  bacillus  from  blood  cultures  during  life, 
which  he  identified  as  B.  aerogenes  capsulatus,  and  which  cor- 
responded to  Achalme's  description  of  his  bacillus ;  neither 
Hewlett  nor  Gwyn  were  certain  in  what  relationships  their 
organisms  stood  to  the  disease.  Foullerton  and  Rist  declare 
that  Achalme's  bacillus  is  identical  to  Klein's  Bac.  enteritidis 
sporogenes  (which  in  turn  is  the  same  as  that  first  described  by 
Welch  in  America  as  Bac.  aerogenes  capsulatus),  and  has  no 
bearing  upon  the  cause  of  acute  rheumatism.  But  the  organism 

1  We  are  indebted  to  Dr.  J.  W.  Eyre  for  the  use  of  this  figure. 


ACUTE   RHEUMATIS 


which  appears  to  have  strongest  claims  is  a  small  diplococcus 
observed  by  Triboulet,  Westphal  and  Wassermann,  Meyer,  and 
Allaria,  the  characters  and  action  of  which  have  been  investi- 
gated especially  by  Poynton  and  Paine.  These  latter  observers 
found  this  organism  in  eight  successive  cases  of  acute  rheuma- 
tism, and  obtained  pure  cultures  both  from  the  blood  during  life 
and  also  from  some  of  the  lesions  after  death  ;  they  also  found 
it,  on  microscopic  examination,  in  all  the  important  lesions  of 
the  disease.  The  organism  is  a  minute  coccus  about  .5  ft  in 
diameter ;  in  the  tissues  it  usually  occurs  in  pairs,  in  fluid 
cultures  it  forms  short  chains,  whilst  on  solid  media  it  is  irregu- 
larly arranged  in  masses.  It  stains  readily  with  the  ordinary 
basic  dyes,  but  loses  the  stain  in  Gram's  method.  For  isolation 
the  best  medium  was  found  to  be  a  mixture  of  bouillon  and 
milk,  rendered  slightly  acid  by  lactic  ^id ;  from  growths  on  this 
medium  sub-cultures  may  be  made  on  blood  agar,  on  which  the 
organism  produces  small  circular,  yellowish-white  colonies,  show- 
ing under  a  low  magnification  a  slightly  granular  appearance. 
On  intravenous  injection  of  pure  cultures  in  rabbits  they  found 
as  results,  polyarthritis  and  synovitis,  valvulitis  and  pericarditis— 
without  any  suppurative  change ;  along  with  these  there  were 
also  marked  symptoms  referable  to  the  lesions  of  the  heart, 
joints,  etc.  These  results  are  of  a  definite  nature,  and  it  remains 
to  be  seen  to  what  extent  they  receive  confirmation  at  the  hands 
of  other  observers,  especially  when  the  experimental  inquiries  are 
made  with  animals  naturally  susceptible  to  disorders  resembling 
human  rheumatism. 

Singer,  as  a  result  of  a  study  of  five  fatal  cases  of  acute 
rheumatism  and  two  of  chorea  rheumatica,  isolated  a  strepto- 
coccus in  pure  culture  from  five  cases ;  streptococcus  and 
staphylococcus  aureus,  in  association,  from  two  cases ;  and 
staphylococcus  aureus,  alone,  from  one  case ;  and  sections  of 
the  various  tissues  and  of  the  cardiac  vegetations  upon  staining 
showed  streptococci  and  diplococci  in  more  or  less  abundance. 
He  believes  that  there  should  not  be  any  claim  allowed  for 
specificity,  such  as  Wassermann,  and  Poynton  and  Paine  hold 
for  their  micrococci,  but  is  of  the  opinion  that  acute  rheuma- 
tism is  only  one  of  the  many  expressions  of  the  variable  activities 
of  the  ordinary  pyogenic  cocci.  Menzer  also  inclines  to  Singer's 
views,  as  a  result  of  a  study  of  two  cases  of  acute  rheumatism, 


204    INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 

wherein  he  isolated  a  streptococcus  which  he  regarded  simply 
as  streptococcus  pyogenes. 

Methods  of  Examination  in  Inflammatory  and  Suppurative 
Conditions.  —  These  are  usually  of  a  comparatively  simple  nature, 
and  include  (i)  microscopic  examination,  (2)  the  making  of 
cultures. 

(1)  The  pus  or  other  fluids  should  be  examined  microscopi- 
cally, first  of  all  by  means  of  film  preparations  in  order  to  deter- 
mine the  characters  of  the  organisms  present.     The  films  should 
be  stained  (a)  by  one  of  the  ordinary  solutions,  such  as  carbol- 
thionin-blue  (p.  101),  or  a  saturated  watery  solution  of  methylene- 
blue ;  and  (b)  by  Gram's  method.     The  use  of  the  latter  is  of 
course  of  high  importance  as  an  aid  in  the  recognition. 

(2)  As  most  of  the  pyogenic  organisms  grow  readily  on  the' 
agar  media  at  37°  C,  pure^cultures  can  be  more  rapidly  obtained 
by  plating  in  the  ordinary  way  than  by  using  gelatin.     When 
the  presence  of  either  pneumococci  or  streptococci  is  suspected, 
this  method  ought  always  to  be  used,  sub-cultures  preferably  at 
first   being    made   in    milk.     Inoculation  experiments    may   be 
carried  out  as  occasion  arises. 

In  cases  of  suspected  blood  infection  the  examination  of  the 
blood  is  to  be  carried  out  by  the  methods  already  described 
(P.  72)- 


CHAPTER   VIII. 

INFLAMMATORY   AND    SUPPURATIVE    CONDITIONS, 
CONTINUED:   THE   ACUTE   PNEUMONIAS. 

Introductory.  —  The  term  Pneumonia  is  applied  to  several 
conditions  which  present  differences  in  pathological  anatomy 
and  in  origin.  All  of  these,  however,  must  be  looked  on  as 
varieties  of  inflammation  in  which  the  process  is  modified  in 
different  ways,  depending  on  the  special  structure  of  the  lung 
or  of  the  parts  which  compose  it.  There  is,  first  of  all,  and,  in 
adults,  the  commonest  type,  the  acute  croupous  or  lobar  pneu- 
monia, in  which  an  inflammatory  process  attended  by  abundant 
fibrinous  exudation  affects,  by  continuity,  the  entire  tissue  of  a 
lobe  or  of  a  large  portion  of  the  lung.  It  departs  from  the 
course  of  an  ordinary  inflammation  in  that  the  reaction  of  the 
connective  tissue  of  the  lung  is  relatively  slight,  and  there  is 
usually  no  tendency  for  organisation  of  the  inflammatory  exuda- 
tion to  take  place.  Secondly,  there  is  the  acute  catarrhal  or 
lobular  pneumonia,  where  a  catarrhal  inflammatory  process 
spreads  from  the  capillary  bronchi  to  the  air  vesicles,  and  in 
these  a  change,  consisting  largely  of  proliferation  of  the  endo- 
thelium  of  the  alveoli,  takes  place  which  leads  to  consolidation 
of  patches  of  the  lung  tissue.  Up  till  1889  acute  catarrhal 
pneumonia  was  comparatively  rare  except  in  children.  In  adults 
it  was  chiefly  found  as  a  secondary  complication  to  some  condi- 
tion such  as  diphtheria,  typhoid  fever,  etc.  Since  the  recent 
epidemics  of  influenza,  however,  it  has  been  of  much  more  fre- 
quent occurrence  in  adults,  has  assumed  a  very  fatal  tendency, 
and  has  presented  the  formerly  quite  unusual  feature  of  being 
sometimes  the  precursor  of  gangrene  of  the  lung.  Besides 
these  two  definite  types  other  forms  also  occur.  Thus  instead 
of  a  fibrinous  material  the  exudation  may  be  of  a  serous  or 
haemorrhagic  or  of  a  purulent  character.  Cases  of  mixed 
fibrinous  and  catarrhal  pneumonia  also  occur,  and  in  the 

205 


206        INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 

catarrhal  there  may  be  great  leucocytic  emigration.  Haemor- 
rhages also  may  occur  here. 

Besides  the  two  chief  types  of  pneumonia  there  is  another 
group  of  cases  which  are  somewhat  loosely  denominated  sep- 
tic pneumonias,  and  which  may  arise  in  two  ways:  (i)  by  the 
entrance  into  the  trachea  and  bronchi  of  discharges,  blood,  etc., 
which  form  a  nidus  for  the  growth  of  septic  organisms ;  these 
often  set  up  a  purulent  capillary  bronchitis  and  lead  to  infection  of 
the  air  cells  and  also  of  the  interstitial  tissue  of  the  lung  ;  (2)  from 
secondary  pyogenic  infection  by  means  of  the  blood  stream  from 
suppurative  foci  in  other  parts  of  the  body.  (See  Chapter  VII., 
pp.  195  et  seq.)  In  these  septic  pneumonias  various  changes, 
resembling  those  found  in  the  other  types,  are  often  seen  round 
the  septic  foci. 

In  pneumonias,  therefore,  there  may  be  present  a  great  variety 
of  types  of  inflammatory  reaction.  We  shall  see  that  with  all  of 
them  bacteria  have  been  found  associated.  Special  importance 
is  attached  to  acute  croupous  pneumonia  on  account  of  its  course 
and  characters,  but  reference  will  also  be  made  to  the  other 
forms. 

Historical.  —  Acute  lobar  pneumonia  for  long  was  supposed  to  be  an  effect 
of  exposure  to  cold ;  but  many  observers  were  dissatisfied  with  this  view 
of  its  etiology.  Not  only  did  cases  occur  where  no  such  exposure  could  be 
traced,  but  it  had  been  observed  that  the  disease  sometimes  occurred  epidem- 
ically, and  was  occasionally  contracted  by  hospital  patients  lying  in  beds 
adjacent  to  those  occupied  by  pneumonia  cases.  Further,  the  sudden  onset 
and  definite  course  of  the  disease  conformed  to  the  type  of  an  acute  infective 
fever;  it  was  thus  suspected  by  some  to  be  due  to  a  specific  infection.  This 
view  of  its  etiology  was  promulgated  by  Friedlander,  whose  results  (published 
in  1882-83)  were  briefly  as  follows.  In  pneumonic  lungs  there  were  cocci, 
adherent  usually  in  pairs,  and  possessed  of  a  definitely  contoured  capsule. 
These  cocci  could  be  isolated  and  grown  on  gelatin,  and  on  inoculation  in 
mice  they  produced  a  kind  of  septicaemia  with  inflammation  of  the  serous 
membranes.  The  blood  and  the  exudation  in  serous  cavities  contained 
numerous  capsulated  diplococci.  Various  criticisms  of  Friedlander's  views 
soon  appeared,  the  chief  being  that  pneumonia  was  not  produced  by  him  in 
animals,  and  there  is  little  doubt  that  many  of  the  organisms  seen  by 
Friedlander  were  really  Fraenkel's  pneumococcus,  to  be  presently  described. 

By  many  observers  it  had  been  found  that  the  sputum  of  healthy  men,, 
when  injected  into  animals,  sometimes  caused  death,  with  the  same  symptoms 
as  in  the  case  of  the  injection  of  Friedlander's  coccus  ;  and  in  the  blood  and 
serous  exudations  of  such  animals  capsulated  diplococci  were  found.  In  fact, 
it  was  thus  first  discovered  by  G.  M.  Sternberg  of  Washington,  in  September, 


CHARACTERS    OF   BACTERIA   OF   PNEUMONIA.  207 

1880,  and  by  Pasteur,  in  December  of  the  same  year.  A.  Fraenkel  found  that 
the  sputum  of  pneumonic  patients  was  much  more  fatal  and  more  constant  in 
its  effects  than  that  of  healthy  individuals.  The  cocci  which  were  found  in 
animals  dead  of  this  "  sputum  septicaemia,11  as  it  was  called,  differed  from 
Friedlander's  cocci  in  several  respects  to  be  presently  studied.  Fraenkel  fur- 
ther investigated  a  few  cases  of  pneumonia,  and  isolated  from  them  cocci 
identical  in  miscroscopic  appearances,  cultures,  and  pathogenic  effects,  with 
those  isolated  in  sputum  septicaemia.  The  most  extensive  investigations  on 
the  whole  question  were  those  of  Weicliselbaum,  published  in  1886.  This 
author  examined  129  cases  of  the  disease,  and  included  in  his  survey  not  only 
acute  croupous  pneumonia,  but  lobular  and  septic  pneumonias.  From  them 
he  isolated  four  groups  of  organisms,  (i)  Diplococcus  pneumonia.  This  he 
described  as  an  oval  or  lancet-formed  coccus,  corresponding  in  appearance 
and  growth  characters  to  Fraenkel's  coccus.  (2)  Streptococcus  pneiimonice . 
This  on  the  whole  presented  similar  characters  to  the  last  but  it  was  more 
vigorous  in  its  growth,  and  could  grow  below  20°  C.,  though  it  preferred  a 
temperature  of  37°  C.  (3)  Staphylococcus  pyogenes  aureus.  (4)  Bacillus 
pneumonia.  This  was  a  short,  rod-shaped  organism,  which  in  Weichselbaum's 
opinion  was  identical  with  Friedlander's  pneumococcus.  Of  these  organisms 
the  diplococcus  pneumoniae  was  by  far  the  most  frequent.  It  also  occurred 
in  all  forms  of  pneumonia.  Next  in  frequency  was  the  streptococcus  pneu- 
moniae, and  lastly  the  bacillus  pneumoniae.  Inoculation  experiments  were  also 
performed  by  Weichselbaum  with  each  of  the  three  characteristic  cocci  he 
isolated.  The  diplococcus  pneumoniae  and  the  streptococcus  pneumoniae  both 
gave  pathogenic  effects  of  a  similar  kind  in  certain  animals. 

The  general  result  of  these  earlier  observations  was  to  estab- 
lish the  occurrence  in  connection  with  pneumonia  of  two  species 
of  organisms,  each  having  its  distinctive  characters,  viz.  :  — 

1.  Fraenkel's  pneumococcus,  which  is  recognised  to  be  identical 
with  the  coccus  of  "  sputum  septicaemia,"  with  Weichselbaum's 
diplococcus  pneumoniae,  and  probably  also  with  his  streptococcus 
pneumoniae. 

2.  Friedlander's  pneumococcus  (now  known  as  Friedlander's 
pneumobacillus),   which    is   almost   certainly  the    same    as    the 
bacillus  pneumoniae  of  Weichselbaum. 

We  shall  use  the  terms  "  Fraenkel's  pneumococcus "  and 
"  Friedlander's  pneumobacillus,"  as  these  are  now  usually  applied 
to  the  two  organisms. 

Microscopic    Characters    of    the   Bacteria    of    Pneumonia. - 
Methods.  —  The  organisms  present  in  acute  pneumonia  can  best 
be  examined  in   film  preparations  made  from  pneumonic  lung 
(preferably  from  a  part  in  a  stage  of  acute  congestion  or  early 
hepatisation),  or  from  the  gelatinous  parts  of  pneumonic  sputum, 


208        INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 

(here  again  preferably  when  such  sputum  is  either  rusty  or 
occurs  early  in  the  disease),  or  in  sections  of  pneumonic  lung. 
Such  preparations  may  be  stained  by  any  of  the  ordinary  weak 

stains,  such  as  a  watery  solu- 
tion of  methylene-blue,  but 
Gram's  method  is  to  be  pre- 
ferred, with  safranin  or  Bis- 
marck-brown as  a  contrast 
stain.  Ziehl-Neelsen  carbol- 
fuchsin  is  also  very  suitable; 
it  is  best  either  to  stain  with 
it  for  only  a  few  seconds,  or 
to  overstain  and  then  decol- 
orise with  alcohol  till  the 
ground  of  the  preparation  is 

FIG.  77.  — Film  preparation  of  pneumonic  just  tinted.       The  Capsules   Can 

sputum,    showing     numerous    pneumococci  ,  .        _,    ,          ,  +-V,    A          1 

(Fraenkel's)  with  unstained  capsules;  some  be  Stained    by  the    metnOQS     al- 

are  arranged  in  short  chains.     Stained  with  ready    described     (p.     IO6).       In 

carbol-fuchsin.     x  icoo. 

such  preparations  as  the  above, 

and  even  in  specimens  taken  from  the  lungs  immediately  after 
death  (as  may  be  quite  well  done  by  means  of  a  hypodermic 
syringe),  putrefactive  and  other  bacteria  may  be  present,  but 
those  to  be  looked  for  are  capsulated  organisms,  which  may 
be  of  either  or  both  of  the  varieties  mentioned. 

( i )  FraenkeVs  Pnenmococcns.  —  This  organism  occurs  in  the 
form  of  a  small  oval  coccus,  about  I  /JL  in  longest  diameter, 
arranged  generally  in  pairs  (diplococci),  but  also  in  chains  of 
four  to  ten  (Fig.  77).  The  free  ends  are  often  pointed  like 
a  lancet,  hence  the  term  diplococcus  lanceolalns  has  also  been 
applied  to  it.  These  cocci  have  round  them  a  capsule,  which, 
in  films  stained  by  ordinary  methods,  usually  appears  as  an 
unstained  halo,  but  is  sometimes  stained  more  deeply  than  the 
ground  of  the  preparation.  This  difference  in  staining  depends, 
in  part  at  least,  on  the  amount  of  decolorisation  to  which  the 
preparation  has  been  subjected.  The  capsule  is  rather  broader 
than  the  body  of  the  coccus,  and  has  a  sharply  defined  external 
margin.  This  organism  takes  up  the  basic  aniline  stains  with 
great  readiness  and  also  retains  the  stain  in  Gram's  method.  It 
is  the  organism  of  by  far  the  most  frequent  occurrence  in  true 
croupous  pneumonia,  and  in  fact  may  be  said  to  be  rarely  absent. 


FRIEDLANDER'S    PNEUMOBACILLUS. 


209 


pneumobacillus, 


(2)  Friedldnders  Pneumobacillus.  —  As  seen  in  the  sputum 
and  tissues,  this  organism  both  in  its  appearance  and  arrange- 
ment, as  also  in  the  presence  of  a  capsule,  somewhat  resembles 
Frae»kel's  pneumococcus,  and 
it  was  at  first  described  as  the 
"pneumococcus."     The  form,  *: 

however,   is   more  of   a  short  .  j? 

rod   shape,  and  it  has   blunt,      -'^ 

rounded  ends;  it  is  also  rather    <•'-.'          *     *V»  ^^   : 

broader  than  Fraenkel's  pneu- 
mococcus.    It  is  now  usually  ^ 
classed    amongst   the    bacilli, 
especially  in  view  of  the  fact             ,           **•"»»«* 
that  in  cultures  elongated  rod 
forms   may    occur  (Fig.    78). 
The    capsule    has    the    same 

,  -          I<IG.   7».  —  f  nediander's   pneumobacillus, 

general  characters  as  that  of  showing  the  variations  in  length,  also  cap- 

Fraenkel's     Organism.       Fried-    sules-     Film   Preparation  from  exudate  in   a 

case  of  pneumonia,     x  1000. 

lander  s  pneumobacillus  stains 

readily  with  the  basic  aniline  stains,  but  loses  the  stain  in  Gram's 
method,  and  is,  accordingly,  coloured  with  the  contrast  stain,  — 

safranin    or    Bismarck-brown, 
as    above    recommended.      A 
valuable    means    is    thus    af- 
forded    of     distinguishing     it 
from    Fraenkel's   pneumococ- 
I    cus    in    microscopic    prepara- 
1   tions. 

/  Friedlander's  organism  is 
much  less  frequently  present  in 
pneumonia  than  Fraenkel's ; 
sometimes  it  is  associated  with 
the  latter,  very  rarely  it  occurs 
alone. 

In  sputum  preparations 
the  capsule  of  both  pneumo- 
cocci  may  not  be  recognisable, 
and  the  same  is  sometimes  true  of  lung  preparations.  This  is 
probably  due  to  changes  which  occur  in  the  capsule  as  the 
result  of  changes  in  the  vitality  of  the  organisms.  Sometimes 


FlG.  79.  —  Fraenkel's  pneumococcus  in 
serous  exudation  at  site  of  inoculation  in  a 
rabbit,  showing  capsules.  Stained  by  Rd. 
Muir's  method.  X  1000. 


210       INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 


in  preparations  stained  by  ordinary  methods  the  difficulty  of 
recognising  the  capsule  when  it  is  present  is  due  to  the  refrac- 
tive index  of  the  fluid  in  which  the  specimen  is  mounted  being 
almost  identical  with  that  of  the  capsule.  This  difficulty  can 
always  be  overcome  by  having  the  groundwork  of  the  prepara- 
tion tinted. 

The  Cultivation  of  FraenkePs  Pneumococcus.  —  It  is  usually 
difficult,  and  sometimes  impossible,  to  isolate  this  coccus  directly 
from  pneumonic  sputum.  On  culture  media  it  has  not  a  vigor- 
ous growth,  and  when  mixed  with  other  bacteria  it  is  apt  to  be 
overgrown  by  the  latter.  To  get  a  pure  culture  it  is  best  to 
insert  a  small  piece  of  the  sputum  beneath  the  skin  of  a  rabbit 
or  a  mouse.  In  about  forty-eight  hours  the  animal  will  die,  with 
numerous  capsulated  pneumococci  throughout  its  blood.  From 
the  heart  blood,  cultures  can  be  easily  obtained. 
Cultures  can  also  be  got  post  mortem  from  the 
lungs  of  pneumonic  patients  by  streaking  a 
number  of  agar  or  blood-agar  tubes  with  a 
scraping  taken  from  the  area  of  acute  con- 
gestion or  commencing  red  hepatisation,  and 
incubating  them  at  37°  C.  The  colonies  of  the 
pneumococcus  appear  as  almost  transparent 
small  discs  which  have  been  compared  to  drops 
of  dew  (Fig.  80).  This  method  is  also  some- 
times successful  in  the  case  of  sputum. 

The  appearances  presented  in  cultures  by 
different  varieties  of  the  pneumococcus  vary 
somewhat.  It  always  grows  best  on  blood 
serum  on  Pfeiffer's  blood  agar  or  in  milk.  It 
usually  grows  well  on  ordinary  agar  or  in 
bouillon,  but  not  so  well  on  glycerin  agar. 
In  a  stroke  culture  on  blood  serum  growth 
appears  as  an  almost  transparent  pellicle  along 
the  track,  with  isolated  colonies  at  the  margin.  On  agar  media 
it  is  more  manifest,  but  otherwise  has  similar  characters.  The 
appearances  are  similar  to  those  of  a  culture  of  streptococcus 
pyogenes,  but  the  growth  is  less  vigorous,  and  is  more  delicate 
in  appearance.  A  similar  statement  also  applies  to  cultures  in 
gelatin  at  22°  C.,  growth  in  a  stab-culture  appearing  as  a  row  of 
minute  points  which  remain  of  small  size ;  there  is,  of  course,  no 


FIG.  80.  —  Stroke- 
culture  of  Fraenkel's 
pneumococcus  o  n 
blood  agar.  The  col- 
onies are  unusually 
large  and  distinct. 
Twenty-four  hours' 
growth  at  37°  C. 
Natural  size. 


CULTIVATION   OF   THE   PNEUMOCOCCUS.  211 

liquefaction  of  the  medium.  On  agar  plates  colonies  are  almost 
invisible  to  the  naked  eye,  but  under  a  low  power  of  the  micro- 
scope appear  to  have  a  compact  finely  granular  centre  and  a  pale 
transparent  periphery.  In  bouillon,  growth  forms  a  slight  tur- 
bidity, which  settles  to  the  bottom  of  the  vessel  as  a  slight  dust- 
like  deposit.  Qn  potatoes,  as  a  rule,  no  growth  appears.  Cultures 
on  such  media  may  be  maintained  for  one  or  two  months,  if  fresh 
sub-cultures  are  made  every  four  or  five  days,  but  they  tend  ulti- 
mately to  die  out.  They  also  rapidly  lose  their  virulence,  so 
that  four  or  five  days  after  isolation  from  an  animal's*  body  their 
pathogenic  action  is  already  diminished.  Eyre  and  Washbourn, 
however,  have  succeeded  in  maintaining  cultures  in  a  condition 
of  constant  virulence  for  at  least  three  months  by  growing  the 
organisms  on  agar  smeared  with  rabbits'  blood.  The  agar  must 
be  prepared  with  Witte's  peptone,  must  not  be  heated  over 
1 00°  C,  and  after  neutralisation  (rosolic  acid  being  used  as  the 
indicator)  must  have  .5  per  cent  of  normal  sodium  hydrate  added. 
The  tubes  when  inoculated  are  to  be  kept  at  37.5°  C.  and  sealed 
to  prevent  evaporation.  In  none  of  the  ordinary  artificial  media 
do  pneumococci  develop  a  capsule,  but  in  milk  cultures  cap- 
sules are  usually  to  be  demonstrated.  They  usually  appear  as 
diplococci,  but  in  preparations  , 

made  from  the  surface  of  agar  V  '  -  ' 

or  from  bouillon,  shorter  or 
longer  chains  may  be  observed 
(Fig.  8 1).  After  a  few  days' 
growth  they  lose  their  regular  f  i  ^  ~i 

shape  and  size,  and  involution  ^.  ,/ 

forms   appear.       Usually   the     - 
pneumococcus  does  not  grow  X,  Wf 

below  22°  C.,  but  forms  in  which         **    ^  «^-    _    ' 
the  virulence  has  disappeared  *^. 

often  grow  well  at  20°  C.     Its 

optimum  temperature  is  37°C.,          FIG. Si.  —  Fraenkel's pneumococcus  from 

•,  .  o  /-i        T     •  a  pure  culture  on  blood  ag[ar  of  twenty-four 

its  maximum  42°  C.     It  is  pre-    „<£„.  growth>  some  in  p3n,  some  in  short 

f  erably  an  aerobe,  but  Can  exist     chains.    Stained  with   weak   carbol-fuchsin. 

without  oxygen.     It  prefers  a 

slightly  alkaline  medium  to  a  neutral,  and  does  not  grow  on  an 
acid  medium.  These  facts  show  that  when  growing  outside  the 
body  on  artificial  media,  the  pneumococcus  is  a  comparatively 


212         INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 


delicate   organism.     There   has    been    described    by   Eyre   and 
Washbourn  a  non-pathogenic  type  of  the  pneumococcus  which 
may  be  found  in  the  healthy  mouth,  and  which  may  also  be 
produced  during  the  saprophytic  growth  of  the 
virulent  form.     From  the  latter  it  differs  generally 
in  its  more  Vigorous  growth,  in  producing  a  uniform 
cloud  in  bouillon,  in  slowly  liquefying  gelatin,  and 
in  growing  on  potato. 

The  Cultivation  of  Friedlander's  Pneumoba- 
cillus.  —  This  organism,  when  present  in  sputum 
or  in  a  pneumonic  lung,  can  be  readily  separated 
by  making  ordinary  gelatin  plate-cultures,  or  a 
series  of  successive  strokes  on  agar  tubes.  The 
surface  colonies  always  appear  as  white  discs, 
which  become  raised  from  the  surface  so  as  to 
appear  like  little  knobs  of  ivory.  From  these, 
pure  cultures  can  be  readily  obtained.  The  ap- 
pearance of  a  stab-culture  in  gelatin  growth  is 
very  characteristic.  At  the  site  of  the  puncture 
there  is  on  the  surface  a  white  growth  heaped 
up,  it  may  be  fully  one-eighth  of  an  inch  above 

3IS 


Crt< 


/>3i 


..j 


*fr  r . 

'••<u-*-' 


^  ^ 

/***:?;••    „   »  **  v  %  *V«*'V 

»*KJT      V  fc      *'       *~  ^  \      Vi*' 

'*  "^   \     JP  \-  £'*  *%»    <- 
i  *^&     A»*v       *"' 


FIG.    82.  —  Stab- 
culture  of    Fried-   the  level  of  the 
lander's      pneumo-    gelatin  ;       along 
bacillus  in  peptone 

gelatin,  showing  the  the  needle  track 

nail-like       appear-    4-uprp  :Q  „   wV>ifP 

Llld.^    lo     CL     W111LC 

ance ;      ten      days 

growth.  Natural  granular  ap- 
pearance,sothat 
the  whole  resembles  a  white 
round-headed  nail  driven  into 
the  gelatin  (Fig.  82).  Hence 
the  name  "nail-like"  which  has  ;  /~V  »  v'^  *  ^  *  »-\  * 
been  applied.  Occasionally  *—  t  ^  .^"^  V^  «* 

bubbles  of  gas  develop  along  •  -»    *  *3P  ^ 

the  line  Of  growth.      There  is  no         Fl°-  83.  — Friedlander's  pneumobacillus.i 

-  .  ..  _         from  a  young  culture  on  agar ;   showing  some 

liquefaction  Of  the  medium.    On    rod-shaped    forms.      Stained    with    thionin- 

sloped    agar  it    forms  a    very  blue-    x  I00°- 

white  growth  with  a  shiny  lustre,  which,  when  touched  with  a 

1  The  apparent  size  of  this  organism,  on  account  of  the  nature  of  its  sheath,  varies 
much  according  to  the  stain  used.  If  stained  with  a  strong  Stain,  e.g.  carbol-fuchsin, 
its  thickness  appears  nearly  twice  as  great  as  is  shown  in  the  figure. 


PNEUMOBACTERIA   IN   OTHER   CONDITIONS.  213 

platinum  needle,  is  found  to  be  of  a  viscous  consistence.  In 
cultures  much  longer  rods  are  formed  than  in  the  tissues  of  the 
body  (Fig.  83).  On  the  surface  si  potatoes  it  forms  an  abundant 
moist  white  layer,  in  which  it  is  usual  to  find  many  small  gas- 
bubbles.  Friedlander's  bacillus  has  active  fermenting  powers  on 
sugars,  though  varieties  isolated  by  different  observers  vary  in 
the  degree  in  which  such  powers  are  possessed.  It  always, 
seems  capable  of  acting  on  dextrose,  lactose,  maltose,  dextrin,, 
and  mannite,  and  sometimes  also  on  glycerin.  The  substances 
produced  by  the  fermentation  vary  with  the  sugar  fermented, 
but  include  ethylic  alcohol,  acetic  acid,  laevolactic  acid,  succinic 
acid,  along  with  hydrogen  and  carbonic  acid  gas.  The  amount 
of  acid  produced  from  lactose  seems  only  exceptionally  sufficient 
to  cause  coagulation  of  milk.  It  is  said  that  this  bacillus  is 
identical  with  an  organism  common  in  sour  milk,  and  also  a 
normal  inhabitant  of  the  human  intestine,  viz.,  the  bacterium 
lactis  aerogenes  of  Escherich.  This  latter  bacillus,  however,  is 
non-pathogenic  and  always  acidifies  and  coagulates  milk  within 
24-48  hours,  with  more  or  less  active  formation  of  gas. 

The  Occurrence  of  the  Pneumobacteria  in  Pneumonia  and  other 
Conditions.  —  Capsulated  organisms  have  been  found  in  every 
variety  of  the  disease — in  acute  croupous  pneumonia,  in  broncho- 
pneumonia,  in  septic  pneumonia.  In  the  great  majority  of  these 
it  is  Fraenkel's  pneumococcus  which,  both  microscopically  and 
culturally,  has  been  found  to  be  present.  Friedlander's  pneumo- 
bacillus  occurs  in  only  about  5  per  cent  of  the  cases.  It  may  be 
present  alone  or  associated  with  Fraenkel's  organism.  In  a  case 
of  croupous  pneumonia  the  pneumococci  are  found  all  through 
the  affected  area  in  the  lung,  especially  in  the  exudation  in  the 
air-cells.  They  also  occur  in  the  pleural  exudation  and  effusion, 
and  in  the  lymphatics  of  the  lung.  The  greatest  number  are 
found  in  the  parts  where  the  inflammatory  process  is  most  recent, 
eg.  in  an  area  of  acute  congestion  in  a  case  of  croupous  pneu- 
monia, and  therefore  such  parts  are  preferably  to  be  selected  for 
microscopic  examination,  and  as  the  source  of  cultures.  Some- 
times there  occur  in  pneumonic  consolidation  areas  of  suppura- 
tive  softening,  which  may  spread  diffusely.  In  such  areas  the 
pneumococci  occur  with  or  without  ordinary  pyogenic  organisms, 
streptococci  being  the  commonest  concomitants.  In  other  cases, 
especially  when  the  condition  is  secondary  to  influenza,  gangrene 


214         INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 

may  supervene  and  lead  to  destruction  of  large  portions  of  the 
lung.  In  these  a  great  variety  of  bacteria,  both  aerobes  and 
anaerobes,  are  to  be  found. 

In  ordinary  broncho-pneumonias  also,  Fraenkel's  pneumo- 
coccus  is  usually  present,  sometimes  along  with  pyogenic  cocci ; 
in  the  broncho-pneumonias  secondary  to  diphtheria  it  may  be 
accompanied  by  the  diphtheria  bacillus,  and  also  by  pyogenic 
cocci ;  in  typhoid  pneumonias  the  typhoid  bacillus  or  the  B.  coli 
may  be  alone  present  or  be  accompanied  by  the  pneumococcus, 
and  in  influenza  pneumonias  the  influenza  bacillus  may  occur. 
In  septic  pneumonias  the  pyogenic  cocci  in  many  cases  are  the 
only  organisms  discoverable,  but  the  pneumococcus  may  also  be 
present  Especially  important,  as  we  shall  see,  from  the  point 
•of  view  of  the  etiology  of  the  disease,  is  the  occurrence  in  other 
parts  of  the  body  of  pathological  conditions  associated  with  the 
presence  of  the  pneumococcus.  By  direct  extension  to  neigh- 
bouring parts  empyema,  pericarditis,  and  lymphatic  enlargements 
in  the  mediastinum  and  neck  may  take  place ;  in  the  first  the 
pneumococcus  may  occur  either  alone  or  with  pyogenic  cocci. 
But  distant  parts  may  be  affected,  for  from  the  blood  stream 
both  in  the  early  and  later  stages  of  pneumonia,  pneumococci 
have  been  isolated  by  Prochaska  and  Cole,  thus  explaining  why 
the  pneumococcus  may  be  found  in  suppurations  and  inflam- 
mations in  various  parts  of  the  body  (subcutaneous  tissue,  peri- 
toneum, joints,  kidneys,  liver,  etc.),  in  otitis  media,  ulcerative 
endocarditis  (p.  197),  and  meningitis.  These  conditions  may 
take  place  either  as  complications  of  pneumonia,  or  they  may 
constitute  the  primary  disease.  The  occurrence  of  meningitis 
is  of  special  importance,  for  next  to  the  lungs  the  meninges 
appear  to  be  the  parts  most  liable  to  attack  by  the  pneumococcus. 
A  large  number  of  cases  have  been  investigated  by  Netter,  who 
gives  the  following  tables  of  the  relative  frequency  of  the  prim- 
ary infections  by  the  pneumococcus  in  man :  — 

(1)  In  adults  — 

Pneumonia  .  .  65.95  per  cent  Empyema  .  .  8.53  per  cent 
Broncho-pneumonia  |  Otitis  .  .  .  2.44  ,, 

Capillary  bronchitis  i       *5    5        "          Endocarditis    .         .         1.22        „ 
Meningitis        .         .         13-00        „          Liver  abscess   .         .         1.22 

(2)  In  children  46  cases  were  investigated.     In  29  the  primary  affection 
was  otitis  media,  in  12  broncho-pneumonia,  in  2  meningitis,  in  i  pneumonia, 
in  i  pleurisy,  in  i  pericarditis. 


EXPERIMENTAL   INOCULATION. 


215 


Thus  in  children  the  primary  source  of  infection  is  in  a  great 
many  cases  an  otitis  media,  and  Netter  concludes  that  infection 
takes  place  in  such  conditions  from  the  nasal  cavities. 

Experimental  Inoculation.  —  The  pneumococcus  of  Fraenkel  is 
pathogenic  to  various  animals,  though  the  effects  vary  somewhat 
with  the  virulence  of  the  race  used.  The  susceptibility  of 
different  species,  as  Gamaleia  has  shown,  varies  to  a  consider- 
able extent.  The  rabbit,  and  especially  the  mouse,  are  very 
susceptible;  the 
guinea-pig,  the 
rat,  the  dog, 
and  the  sheep 
occupy  an  inter- 
mediate posi- 
tion; the  pigeon 
is  immune.  In 
the  more  sus- 
ceptible animals 
the  general 
type  of  the  dis- 
ease produced 
is  not  p  n  e  u- 
monia,  but  a 
general  septiccz- 
mia.  Thus,  if 
a  rabbit  or  a 

• 


i  •  FIG.  84.  —  Capsulated  pneumococci  in  blood  taken  from  the 

heart  of  a  rabbit,  dead  after  inoculation  with    pneumonic  sputum. 


jected    simul-    Dried  film,  fixed  with  corrosive  sublimate.     Stained  with  carbol- 
»  .  ,      fuchsin  and  partly  decolorised.     X  1000. 

taneously    with 

pneumonic  sputum,  or  with  a  scraping  from  a  pneumonic  lung, 
death  occurs  in  from  twenty-four  to  forty-eight  hours.  There  is 
some  fibrinous  infiltration  at  the  point  of  inoculation,  the  spleen 
is  often  enlarged  and  firm,  and  the  blood  contains  capsulated 
pneumococci  in  large  numbers  (Fig.  84).  If  the  seat  of  inocula- 
tion be  in  the  lung,  there  generally  results  pleuritic  effusion  on 
both  sides,  and  in  the  lung  there  may  be  a  process  somewhat 
resembling  the  early  stage  of  acute  croupous  pneumonia  in  man. 
There  are  often  also  pericarditis  and  enlargement  of  spleen. 
We  have  already  stated  that  cultures  of  the  pneumococci  on 
artificial  media  in  a  few  days  begin  to  lose  their  virulence. 


2l6         INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 

Now,  if  such  a  partly  attenuated  culture  be  injected  sub- 
cutaneously  into  a  rabbit,  there  is  greater  local  reaction ; 
pneumonia,  with  exudation  of  lymph  on  the  surface  of  the 
pleura,  and  a  similar  condition  in  the  peritoneum,  may  occur. 
In  sheep  greater  immunity  is  marked  by  the  occurrence,  after 
subcutaneous  inoculation,  of  an  enormous  local  sero-fibrinous 
exudation,  and  by  the  fact  that  few  pneumococci  are  found  in 
the  blood  stream.  Intrapulmonary  injection  in  sheep  is  fol- 
lowed by  a  typical  pneumonia,  which  is  generally  fatal.  The 
dog  is  still  more  immune;  in  it  also  intrapulmonary  injection  is 
followed  by  a  fibrinous  pneumonia,  which  is  only  sometimes  fatal. 
Inoculation  by  inhalation  appears  only  to  have  been  performed  in 
the  susceptible  mouse  and  rabbit;  here  also  septicaemia  resulted. 

The  general  conclusion  to  be  drawn  from  these  experiments 
thus  is  that  in  highly  susceptible  animals  virulent  pneumococci 
produce  a  general  septicaemia ;  whereas  in  more  immune  species 
there  is  an  acute  local  reaction  at  the  point  of  inoculation,  and 
if  the  latter  be  in  the  lung,  then  there  may  result  pneumonia, 
which,  of  course,  is  merely  a  local  acute  inflammation  occurring 
in  a  special  tissue,  but  identical  in  essential  pathology  with  an 
inflammatory  reaction  in  any  other  part  of  the  body.  When  a 
dose  of  pneumococci  sufficient  to  kill  a  rabbit  is  injected  sub- 
cutaneously  in  the  human  subject,  it  gives  rise  to  a  local  inflam- 
matory swelling  with  redness  and  slight  rise  of  temperature,  all 
of  which  pass  off  in  a  few  days.  It  is  therefore  justifiable  to 
suppose  that  man  occupies  an  intermediate  place  in  the  scale  of 
susceptibility,  probably  between  the  dog  and  the  sheep,  and  that 
when  the  pneumococcus  gains  an  entrance  to  his  lungs,  the 
local  reaction  in  the  form  of  pneumonia  occurs. 

Analogies  to  the  facts  just  stated  are  afforded  in  the  case 
of  other  diseases  caused  by  bacteria.  Thus,  for  example,  the 
anthrax  bacillus  produces  in  the  human  subject  more  marked 
inflammatory  reaction,  and  is  more  restricted  to  the  local 
lesions,  than  in  the  much  more  susceptible  guinea-pig,  in  which 
it  produces  a  rapidly  fatal  septicaemia.  An  analogous  result  is 
also  obtained  when,  instead  of  taking  animals  of  different  sus- 
ceptibility, the  same  species  of  animal  is  used,  but  the  virulence 
of  the  organism  is  altered ;  for  example,  a  streptococcus,  as 
already  stated,  producing  at  one  time  an  erysipelatous  condition, 
causes  an  acute  septicaemia  when  its  virulence  is  increased. 


EXPERIMENTAL   INOCULATION. 


217 


The  occurrence  in  the  lung  of  inflammatory  conditions  due 
to  other  causes  does  not  make  it  less  likely  that  the  great  major- 
ity of  cases  of  acute  pneumonia  which  occur  under  natural  con- 
ditions have  as  the  causal  agent  the  pneumococcus.  For  in  the 
latter  we  have  an  organism  with  certain  very  definite  micro- 
scopic and  biological  characters,  which  is  certainly  present  in 
the  great  majority  of,  if  not  in  all,  cases  of  the  disease.  Its 
action  as  a  producer  of  general  septicaemia  in  animals,  we  have 
seen,  finds  a  perfectly  rational  explanation  in  the  different 
degrees  of  susceptibility  which  exist  towards  it  in  different 
species.  In  this  connection  the  occurrence  of  manifestations  of 
general  infection  associated  with  pneumonia  in  man  is  of  the 
highest  importance.  We  have  seen  that  meningitis  and  other 
inflammations  are  not  very  rare  complications  of  the  disease, 
and  such  cases  form  a  link  connecting  the  local  disease  in  the 
human  subject  with  the  general  septicaemic  processes  which  may 
be  produced  artificially  in  the  more  susceptible  representatives 
of  the  lower  animals. 

A  fact  which  has,  in  the  minds  of  some,  rather  militated 
against  the  pneumococcus  being  the  cause  of  pneumonia,  is  the 
discovery  of  this  organism  in  the  saliva  of  healthy  men.  This 
fact  was  early  pointed  out  by  Pasteur,  Sternberg,  and  also  by 
Fraenkel,  and  their  observations  have  been  confirmed  by  many 
other  observers.  It  can  certainly  be  isolated  from  the  mouths 
of  a  considerable  proportion  of  normal  men,  from  their  nasal 
cavities,  etc.,  being  probably  in  any  particular  individual  more 
numerous  at  some  times  than  at  others,  and  sometimes  being 
entirely  absent.  This  can  be  proved,  of  course,  by  inoculation 
of  susceptible  animals.  Such  a  fact,  however,  does  not  neces- 
sarily imply  that  the  pneumococcus  is  not  the  cause  of  pneu- 
monia. It  only  indicates  the  importance  of  predisposing  causes 
in  the  etiology  of  the  disease,  and  it  is  further  to  be  observed 
that  we  have  corresponding  facts  in  the  case  of  the  diseases 
caused  by  pyogenic  staphylococci,  streptococci,  the  bacillus  coli, 
etc.  It  is  probable  that  by  various  causes  the  vitality  and  power 
of  resistance  of  the  lung  are  diminished,  and  that  then  the  pneu- 
mococcus gains  an  entrance.  In  relation  to  this  possibility  we 
have  the  very  striking  facts  that  in  the  irregular  forms  of  pneu- 
monia, secondary  to  such  conditions  as  typhoid  and  diphtheria, 
the  pneumococcus  is  very  frequently  present,  alone  or  with 


218         INFLAMMATORY   AND    SUPPURATIVE   CONDITIONS. 

other  organisms.  Apparently  the  effects  produced  by  such 
bacteria  as  the  B.  typhosus  and  the  B.  diphtherias  can  devitalize 
the  lung  to  such  an  extent  that  secondary  infection  by  the  pneu- 
mococcus  is  more  likely  to  occur  and  set  up  pneumonia.  We 
can  therefore  understand  how  much  less  definite  devitalising 
agents  such  as  cold,  alcoholic  excess,  etc.,  can  play  an  important 
part  in  the  causation  of  pneumonia.  In  this  way  also  other 
abnormal  conditions  of  the  respiratory  tract,  a  slight  bronchitis, 
etc.,  may  play  a  similar  part. 

It  is  more  difficult  to  explain  why  sometimes  the  pneumo- 
coccus  is  associated  with  a  spreading  inflammation,  as  in  croupous 
pneumonia,  whilst  at  other  times  it  is  localised  to  the  catarrhal 
patches  in  broncho-pneumonia.  It  is  quite  likely  that  in  the 
former  condition  the  organism  is  possessed  of  a  different  order 
of  virulence,  though  of  this  we  have  no  direct  proof.  We  have, 
however,  a  closely  analogous  fact  in  the  case  of  erysipelas,  which, 
we  have  stated  reasons  for  believing,  is  produced  by  a  strepto- 
coccus which,  when  less  virulent,  causes  only  local  inflammatory 
and  suppurative  conditions. 

Summary. — We  may  accordingly  summarise  the  facts  re- 
garding the  relation  of  Fraenkel's  pneumococcus  to  the  disease 
by  saying  that  it  can  be  isolated  from  nearly  all  cases  of  acute 
croupous  pneumonia,  and  also  from  a  considerable  proportion 
of  other  forms  of  pneumonia.  When  injected  into  the  lungs  of 
moderately  insusceptible  animals  it  gives  rise  to  pneumonia.  If, 
in  default  of  the  crucial  experiment  of  intrapulmonary  injection 
in  the  human  subject,  we  take  into  account  the  facts  we  have 
discussed,  we  are  justified  in  holding  that  it  is  the  chief  factor  in 
causing  croupous  pneumonia,  and  also  plays  an  important  part 
in  other  forms.  Pneumonia,  in  the  widest  sense  of  the  term,  is, 
however,  not  a  specific  affection,  and  various  inflammatory  con- 
ditions in  the  lungs  can  be  set  up  by  the  different  pyogenic 
organisms,  by  the  bacilli  of  diphtheria,  of  influenza,  etc. 

The  possibility  of  Friedlander's  pneumobacillus  having  an 
etiological  relationship  to  pneumonia  has  been  much  disputed. 
Its  discoverer  found  that  it  was  pathogenic  towards  mice  ancl 
guinea-pigs,  and  to  a  less  extent  towards  dogs.  Rabbits  appeared 
to  be  immune.  The  type  of  the  disease  was  of  the  nature  of  a 
septicaemia.  No  extended  experiments,  such  as  those  performed 
by  Gamaleia  with  Fraenkel's  coccus,  have  been  done,  and  there- 


TOXINS    OF   THE    PNEUMOCOCCUS.  219 

fore  we  cannot  say  whether  any  similar  pneumonic  effects  are 
produced  by  it  in  partly  susceptible  animals.  The  organism 
appears  to  be  present  alone  in  a  small  number  of  cases  of 
pneumonia,  and  the  fact  that  it  also  appears  to  have  been  the 
only  organism  present  in  certain  septicaemic  complications  of 
pneumonia,  such  as  empyema  and  meningitis,  render  it  possible 
that  it  may  be  the  causal  agent  in  a  few  cases  of  the  disease. 

In  the  septic  pneumonias  the  different  pyogenic  organisms 
already  described  are  found,  and  sometimes  in  ordinary  pneu- 
monias, especially  the  catarrhal  forms,  other  organisms,  such  as 
the  B.  coli  or  its  congeners,  may  be  the  causal  agent. 

The  Toxins  of  FraenkePs  Pneumococcus.  —  Pneumonia  in  its 
commonest  types  is  a  disease  which  presents  in  many  respects 
the  characters  of  an  acute  poisoning.  In  very  few  cases  does 
death  take  place  from  the  functions  of  the  lungs  being  interfered 
with  to  such  an  extent  as  to  cause  asphyxia.  It  is  from  car- 
diac failure,  from  grave  interference  with  the  heat-regulating 
mechanism,  and  from  a  general  nervous  depression  that  death 
usually  results.  These  considerations,  taken  in  connection  with 
the  fact  that  in  man  the  pneumococci  are  usually  confined  to 
the  lung,  suggest  that  they  may  produce  their  general  effects  by 
means  of  toxins.  The  subject  has  been  investigated  by  Em- 
merich and  Fowitsky  and  by  G.  and  F.  Klemperer.  The  latter 
isolated  from  recent  bouillon  cultures,  by  the  methods  of  Brie- 
ger  and  Fraenkel  (p.  172),  bodies  having  the  reactions  of  the 
toxalbumins  obtained  in  the  case  of  other  bacteria.  When 
injected,  these  toxalbumins  (which  they  called  "  pneumotoxin  ") 
produced  symptoms  in  rabbits,  and  when  they  were  derived  not 
from  bouillon  cultures  but  from  the  blood  of  animals  dead  of  the 
disease,  they  could  produce  fatal  effects.  This  work  was  done 
before  media  had  been  devised  on  which  the  pneumococcus  can 
live  for  a  number  of  weeks,  and  therefore  only  the  toxins 
resulting  from  a  few  days'  growth  were  used.  Of  the  nature  of 
the  poisons  which  were  obtained  we  know  nothing.  Carnot 
describes  a  toxin  which,  when  introduced  into  an  animal's  lung, 
gave  rise  to  pneumonic  conditions,  and  also  secondarily  produced 
changes  in  the  heart  and  symptoms  of  cardiac  affection  similar 
to  those  occurring  during  the  disease  in  the  human  subject. 

Immunisation  against  the  Pneumococcus.  —  Animals  can  be 
immunised  against  the  pneumococcus  either  by  inoculation  with 


220         INFLAMMATORY   AND    SUPPURATIVE    CONDITIONS. 

attenuated  cultures  or  by  the  injection  of  toxic  bodies  derived 
from  cultures.  The  former  can  be  effected  by  cultures  which 
have  become  attenuated  by  growth  on  artificial  media,  or  by  the 
naturally  attenuated  cocci  which  occur  in  the  sputum  after  the 
crisis  of  the  disease.  Netter  effected  immunisation  by  injecting 
an  emulsion  of  the  dried  spleen  of  an  animal  dead  of  pneumo- 
coccus  septicaemia.  Here  the  cocci  were  attenuated  by  the 
drying.  Virulent  cultures  killed  by  heating  at  62°  C.  have  also 
been  used,  immunisation  being  here  accomplished  by  the  intra- 
cellular  toxins.  The  Klemperers  found  that  injection  of  rusty 
sputum  kept  at  60°  C.  for  one  to  two  hours  and  then  filtered, 
and  of  filtered  or  unfiltered  bouillon  cultures  similarly  treated, 
had  a  like  result.  In  all  cases  one  or  two  injections  of  the 
modified  bacteria  or  toxin  were  sufficient  for  immunisation.  It 
was  three  days  in  the  case  of  intravenous  injection,  and  fourteen 
days  in  the  case  of  subcutaneous  injection,  before  immunity  was 
established,  and  the  latter  laste'd  a  month  or  more.  The  im- 
munity was  accompanied  by  the  development  in  the  blood  of 
antitoxic  substances  which  had  no  effect  either  outside  or  inside 
the  body  in  killing  the  pneumococci,  but  merely  neutralised 
their  toxins.  Such  antitoxins  not  only  protected  a  rabbit  against 
subsequent  inoculation  with  pneumococci,  but  if  injected  within 
twenty-four  hours  after  inoculation,  prevented  death.  A  pro- 
tective serum  has  also  been  obtained  by  Washbourn,  who,  as 
already  described,  has  succeeded  in  obtaining  pneumococcus 
cultures  of  constant  virulence.  This  observer  immunised  a  pony 
by  using  (i)  broth  cultures  killed  by  one  hour's  exposure  to  60° 
C. ;  (2)  living  agar  cultures;  (3)  living  broth  cultures.  From 
this  animal  a  serum  of  high  protective  power  was  obtained.  It 
protected  susceptible  animals  against  many  times  an  otherwise 
fatal  dose,  and  it  also  had  a  curative  action,  only,  however,  when 
injected  very  soon  after  inoculation.  To  what  the  protective 
properties  of  such  sera  are  due  requires  further  investigation. 
In  this  connection  an  interesting  fact  observed  by  Mennes  may 
be  noted,  namely,  that  normal  leucocytes  only  become  phagocytic 
towards  pneumococci  when  they  are  lying  in  the  serum  of  an 
animal  immunised  against  this  bacterium. 

If  in  these  sera  antitoxins  are  present  this  may  shed  new 
light  on  what  occurs  in  man  in  the  case  of  recovery  from 
pneumonia.  The  view  has  been  advanced  that  the  crisis  so 


METHODS   OF   EXAMINATION.  221 

characteristic  of  a  non-fatal  case  of  the  disease  takes  place  when 
the  balance  of  antitoxin  against  toxin  is  in  favour  of  the  former. 
The  pneumococci  after  the  crisis,  as  has  been  proved  both 
culturally  and  by  inoculation  experiments,  are  still  vital  and 
virulent,  though  not  so  virulent  as  when  the  fever  is  at  its  height. 
On  them  directly  the  antitoxin  has  no  effect,  but  any  toxin  now 
elaborated  by  them  is  neutralised,  and  has  no  longer  either  local 
or  general  pathogenic  effects. 

A  fact  interesting  as  corroborating  the  view  that  the  pneumo- 
coccus  is  really  the  cause  of  acute  lobar  pneumonia  is  that  the 
serum  of  patients  who  have  recovered  from  pneumonia  has 
in  a  certain  proportion  of  cases  a  protective  effect  against  the 
pneumococcus  in  rabbits.  So  far  as  our  knowledge  goes,  such 
a  protective  serum  is  specific,  or,  in  other  words,  protects  only 
against  the  organism  by  the  action  of  which  its  protective 
properties  have  been  produced,  and  therefore  it  must  be  against 
the  pneumococcus  that  the  human  subject  requires  protection 
in  pneumonia. 

The  Klemperers  treated  a  certain  number  of  cases  of  human 
pneumonia  by  serum  derived  from  immune  animals,  and  appar- 
ently with  a  certain  measure  of  success,  and  Washbourn's  serum 
has  also  been  used.  Although  the  use  of  these  sera  apparently 
causes  the  temperature  to  fall,  and  in  some  cases  appears  to 
hasten  a  crisis,  further  experience  is  necessary  before  their  value 
in  therapeutics  can  be  properly  estimated. 

If  a  small  amount  of  a  culture  of  Fraenkel's  pneumococcus 
be  placed  in  an  anti-pneumococcic  serum,  an  aggregation  of  the 
bacteria  into  clumps  occurs.  Such  an  agglutination,  as  it  is 
called,  is  frequently  observed  under  similar  circumstances  with 
other  bacteria.  The  phenomenon  is  not  invariably  associated 
with  the  presence  of  protective  bodies  in  a  serum,  but  it  has 
been  used  for  diagnostic  purposes  in  the  differentiation  of  sore 
throats  due  to  pneumococcus  infection  from  those  due  to  other 
bacteria.  Whether  the  method  is  reliable  has  still  to  be  proved. 

Methods  of  Examination.  —  These  have  been  already  de- 
scribed, but  may  be  summarised  thus:  (i)  Microscopic.  Stain 
films  from  the  densest  part  of  the  sputum  or  from  the  area  of 
spreading  inflammation  in  the  lung  by  Gram's  method  and  by 
carbol-fuchsin,  etc.  (p.  102),  in  the  latter  case  without  decoloris- 
ing the  groundwork  of  the  preparation. 


222       INFLAMMATORY   AND    SUPPURATIVE    CONDITIONS. 

(2)  By  cultures,  (a)  FraenkeFs  pneumococcus.  With  similar 
material  make  successive  strokes  on  agar,  blood  agar,  or  blood 
serum,  or  by  inoculation  of  milk  tubes.  The  most  certain 
method,  however,  is  to  inject  some  of  the  material  containing 
the  suspected  cocci  into  a  rabbit.  If  the  pneumococcus  be 
present  the  animal  will  die,  usually  within  forty-eight  hours,  wrth 
numerous  capsulated  pneumococci  in  its  heart  blood.  With  the 
latter  inoculate  tubes  of  the  above  media  and  observe  the  growth. 
In  some  cases  of  severe  pneumococcic  infection  the  organism 
may  be  cultivated  from  the  blood  obtained  by  venesection 
(P-  73)-  (b)  Friedldnder1  s  pneumobacilhis  can  be  readily  isolated 
either  by  ordinary  agar  and  gelatin  plates  or  by  successive  strokes 
on  agar  media. 


CHAPTER   IX. 

GONORRHCEA,   SOFT   SORE,   SYPHILIS. 
GONORRHCEA. 

Introductory.  —  The  micrococcus  now  known  to  be  the  cause 
of  gonorrhoea,  and  now  called  the  gonococcus,  was  first  described 
by  Neisser,  who  in  1879  gave  an  account  of  its  microscopical 
characters  as  seen  in  the  pus  of  gonorrhoeal  affections,  both  of 
the  urethra  and  of  the  conjunctiva.  He  considered  that  this 
organism  was  peculiar  to  the  disease,  and  that  its  characters 
were  distinctive.  Later  it  was  successfully  isolated  and  culti- 
vated on  solidified  blood  serum  by  Bumm  and  others.  Its 
characters  have  since  been  minutely  studied,  and  by  inoculations 
of  cultures  on  the  human  subject  its  causal- relationship  to  the 
disease  has  been  conclusively  established. 

The  Gonococcus.  —  Microscopical  Characters.  —  The  organism 
of  gonorrhoea  is  a  small  micrococcus  which  usually  is  seen  in  the 
diplococcus  form,  the  adjacent  margins  of  the  two  cocci  being 
flattened,  or  even  slightly  concave,  so  that  between  them  there  is 
a  small  oval  interval  which  does  not  stain.  An  appearance  is 
thus  presented  which  has  been  compared  to  that  of  two  beans 
placed  side  by  side  (vide  Fig.  85).  When  division  takes  place 
in  the  two  members  of  a  diplococcus  a  tetrad  is  formed,  which, 
however,  soon  separates  into  two  sets  of  diplococci  —  that  is  to 
say,  arrangement  as  diplococci  is  much  commoner  than  as  tetrads. 
Cocci  in  process  of  degeneration  are  seen  as  spherical  elements 
of  various  size,  some  being  considerably  swollen. 

These  organisms  are  found  in  large  numbers  in  the  pus  of 
acute  gonorrhoea,  both  in  the  male  and  female,  and  for  the  most 
part  are  contained  within  the  leucocytes.  In  the  earliest  stage, 
when  the  secretion  is  glairy,  a  considerable  number  are  lying 
free,  or  are  adhering  to  the  surface  of  desquamated  epithelial 
cells,  but  when  it  becomes  purulent  the  large  proportion  within 

223 


224  GONORRHCEA,    SOFT    SORE,   SYPHILIS. 

• 

leucocytes  is  a  very  striking  feature.  In  the  leucocytes  they 
lie  within  the  protoplasm,  especially  superficially,  and  are  often 
so  numerous  that  the  leucocytes  appear  to  be  filled  with  them, 
and  their  nuclei  are  obscured.  As  the  disease  becomes  more 
chronic,  the  gonococci  gradually  become  diminished  in  number, 
though  even  in  long-standing  cases  they  may  still  be  found  in 
considerable  numbers.  They  are  also  present  in  the  purulent 
secretion  of  gonorrhoeal  conjunctivitis,  also  in  various  parts  of 
the  female  genital  organs  when  these  parts  are  the  seat  of  true 
gon6rrhoeal  infection,  and  they  have  been  found  in  some  cases 

in  the  secondary  infections 
of  the  joints  in  the  disease, 
as  will  be  described  below. 

Staining.  —  The  gonococ- 
cus  stains  readily  and  deeply 
with  a  watery  solution  of  any 
of  the  basic  aniline  dyes  — 
methylene-blue,  fuchsin,  etc. 
It  is,  however,  easily  decol- 
prised,  and  it  completely 
loses  the  stain  by  Gram's 
method  —  an  important  point 
in  the  microscopical  examina- 

FlG.  85.  —  Portion  of  film  of  gonorrhoeal 
pus,  showing  the  characteristic  arrangement  of     tlOn. 
the  gonococci  within  leucocytes.  Cultivation  Of  the  GonOCOC- 

Stained  with  fuchsin.     X  1000. 

cus.  —  This  is  attended  with 

some  difficulty,  as  the  suitable  media  and  conditions  of  growth 
are  somewhat  restricted.  The  most  suitable  media  are  solidified 
blood  serum  (especially  human  serum  and  rabbit's  serum), 
blood  agar,  and  Wertheim's  medium,  which  consists  of  one 
part  of  fluid  serum  added  to  two  parts  of  liquefied  agar  at  a 
temperature  of  45°  C.  and  then  allowed  to  solidify  by  cooling. 
The  serum  may  be  obtained  from  the  blood  of  the  human  pla- 
centa ;  pleuritic,  hydrocele,  or  other  effusion  may  also  be  used. 
Growth  takes  place  best  at  the  temperature  of  the  body,  and 
ceases  altogether  at  25°  C.  Cultures  are  obtained  by  taking 
some  pus  on  the  loop  of  the  platinum  needle  and  inoculating 
one  of  the  media  mentioned  by  leaving  minute  quantities  here 
and  there  on  the  surface.  The  medium  may  be  used  either  as 
ordinary  "  sloped  tubes "  or  as  a  thin  layer  in  a  Petri's  dish. 


CULTIVATION   OF   THE   GONOCOCCUS.  225 

The  young  colonies  are  visible  within  forty-eight  hours,  and  often 
wkhin  twenty-four  hours.  They  appear  around  the  points  of 
inoculation  as  small  semi-transparent  discs  of  irregularly  rounded 
shape,  the  margin  being  undulated  and  sometimes  showing  small 
processes.  The  colonies  vary  somewhat  in  size  and  tend  to 
remain  more  or  less  separate.  They  generally  reach  their 
maximum  size  on  the  fourth  or  fifth  day,  and  are  usually  found 
to  be  dead  on  the  ninth  day,  often  much  earlier.  On  the 
medium  of  Wertheim  the  period  of  active  growth  and  the 
duration  of  life  are  somewhat  longer.  Even  if  impurities  are 
present,  pure  sub-cultures  can  generally  be  obtained  by  the  above 
methods  from  colonies  of  the  gonococcus  which  may  be  lying 
separate.  In  the  early  stage  of  the  disease  the  organism  is 
present  in  the  male  urethra 
in  practically  pure  condition, 
and  if  the  meatus  of  the 
urethra  be  sterilised  by  wash- 
ing with  weak  solution  of 
corrosive  sublimate  and  then 
with  absolute  alcohol,  and  5^. 
the  material  for  inoculation 
be  expressed  from  the  deeper 
part  of  the  urethra,  cultures  ,  -^ 

may  often  be  obtained  which  "  •«. 

are  pure  from  the  first.     By  *  *  *  .    " 

successive     sub-cultures     at 

Short    intervals,    growth    may  FlG.86.-Gonococci,  from  a  pure  culture 

'  J      on   blood  agar   ot   twenty-four   hours'  growth. 

be  maintained  indefinitely,  Some  already  are  beginning  to  show  the  swollen 
and  the  organism  gradually  appearance  common  in  older  cultures. 

Stained  with  carbol-thionm-blue.      X  1000. 

flourishes    more    luxuriantly. 

In  culture  the  organisms  have  similar  microscopic  characters  to 
those  described  (Fig.  86),  but  show  a  remarkable  tendency  to 
undergo  degeneration,  becoming  swollen  and  of  various  sizes, 
and  staining  very  irregularly.  Degenerated  forms  are  seen 
even  on  the  second  day,  whilst  in  a  culture  four  or  five  days 
old  comparatively  few  normal  cocci  may  be  found.  The  less 
suitable  the  medium  the  more  rapidly  does  degeneration  take 
place. 

On  ordinary  agar  and  on  glycerin  agar  growth  does  not  take 
place,  or  is  so  slight  that  these  media  are  quite  unsuitable  for 
Q 


226  GONORRHCEA,    SOFT    SORE,    SYPHILIS. 

purposes  of  culture.     The  organism  does  not  grow  on  gelatin,1 
potato,  etc. 

Plate-cultures.  —  The  following  ingenious  method  of  plate-culture  was 
introduced  by  Wertheim  for  the  culture  of  the  gonococcus.  The  medium  of 
culture  is  a  mixture  of  human  blood  serum  and  of  ordinary  agar  (2  per  cent) 
in  equal  parts.  The  serum,  in  a  fluid  and  sterile  condition,  is  put  in  suitable 
quantities  into  two  or  three  test-tubes  and  brought  to  a  temperature  of  45°  C. 
These  are  then  successively  inoculated  with  the  pus  or  other  material  in  the 
same  manner  as  gelatin  tubes  for  ordinary  plates  (vide  p.  54).  To  each  tube 
is  added  an  equal  part  of  ordinary  agar  which  has  been  thoroughly  liquefied 
by  heating  and  allowed  to  cool  also  to  45°  C.  The  mixture  is  then  thoroughly 
shaken  up  and  quickly  poured  out  on  a  plate  or  Petri's  dish  and  allowed  to 
solidify,  the  plates  being  then  incubated  at  a  temperature  of  37°  C.  The 
colonies  of  the  gonococcus  are  just  visible  in  twenty-four  hours,  and  are  seen 
both  in  the  substance  of  the  medium  and  on  the  surface.  The  deep  colonies 
when  examined  with  a  lens  are  minute  and  slightly  nodulated  spheres,  some- 
times showing  little  processes,  whilst  those,  on  the  surface  are  thin  discs  of 
larger  diameter  with  wavy  margin  and  rather  darker  centre.  In  this  way  the 
gonococcus  may  be  separated  from  fluids  which  are  contaminated  with  a  con- 
siderable number  of  other  organisms. 

Relations  to  the  Disease.  —  The  gonococcus  is  invariably 
present  in  the  urethral  discharge  in  gonorrhoea,  and  also  in 
other  parts  of  the  genital  tract  when  these  are  the  seat  of  true 
gonorrhoeal  infection.  Its  presence  in  these  different  positions 
has  been  demonstrated  not  only  by  microscopic  examination 
but  also  by  culture.  From  the  description  of  the  conditions  of 
growth  in  culture,  it  will  be  seen  that  a  life  outside  the  body 
in  natural  conditions  is  practically  impossible  —  a  statement 
which  corresponds  with  the  clinical  fact  that  the  disease  is 
always  transmitted  directly  by  contagion.  Inoculations  of  pure 
cultures  on  the  urethra  of  lower  animals,  and  even  of  apes,  is 
followed  by  no  effect,  but  a  similar  statement  can  be  made  with 
regard  to  inoculations  of  gonorrhoeal  pus  itself.  In  fact,  hith- 
erto it  has  been  found  impossible  to  reproduce  the  disease  by 
any  means  in  the  lower  animals.  On  a  considerable  number  of 
occasions  inoculations  of  pure  cultures  have  been  made  on  the 
human  urethra,  both  in  the  male  and  female,  and  the  disease, 
with  all  its  characteristic  symptoms,  has  resulted.  (Such 

1  Turro  has  announced  that  he  has  cultivated  the  gonococcus  on  acid  gelatin, 
i.e.  ordinary  peptone  gelatin  which  has  not  been  neutralised.  We  have  failed  to  ob- 
tain any  growth  of  the  gonococcus  on  this  medium,  even  when  inoculation  was  made 
from  a  vigorous  growth  on  blood  agar. 


DISTRIBUTION    IN    THE    TISSUES.  22/ 

experiments  have  been  performed  independently  by  Bumm, 
Steinschneider,  Wertheim,  and  others.)  The  causal  relationship 
of  the  organism  to  the  disease  has  therefore  been  completely 
established,  and  it  is  interesting  to  note  how  the  conditions  of 
growth  and  the  pathogenic  effects  of  the  organism  agree  with 
the  characters  of  the  natural  disease. 

Intraperitoneal  injections  of  pure  cultures  of  the  gonococcus  in  white  mice 
produce  a  localised  peritonitis  with  a  small  amount  of  suppuration,  the 
organisms  being  found  in  large  numbers  in  the  leucocytes  (Wertheim).  They 
also  penetrate  the  peritoneal  lining  and  are  found  in  the  sub-endotheliai 
connective  tissue,  but  they  appear  to  have  little  powrer  of  proliferation,  they 
soon  disappear,  and  the  inflammatory  condition  does  not  spread.  Injection 
of  pure  cultures  into  the  joints  of  rabbits,  dogs,  and  guinea-pigs  causes  an 
acute  inflammation,  which,  however,  soon  subsides,  whilst  the  gonococci 
rapidly  die  out :  a  practically  similar  result  is  obtained  when  dead  cultures 
are  used.  These  experiments  show  that  while  the  organism,  when  present  in 
large  numbers,  can  produce  a  certain  amount  of  inflammatory  change  in  these 
animals,  it  has  little  or  no  power  of  multiplying  and  spreading  in  their  tissues. 

Toxin  of  the  Gonococcus.  —  De  Christmas  has  cultivated  the  gonococcus 
in  a  mixture  of  part  of  ascitic  fluid  and  three  parts  of  bouillon,  and  has  found 
that  the  fluid  after  twelve  days1  growth  has  toxic  properties.  At  this  period 
all  the  organisms  are  dead;  such  a  fluid  constitutes  the  "toxin."  The  toxic 
substances  are  precipitated  along  with  the  proteids  by  alcohol,  and  the 
precipitate  after  being  desiccated  possesses  the  toxic  action.  In  young 
rabbits  injection  of  the  toxin  produces  suppuration ;  this  is  well  seen  in  the 
anterior  chamber  of  the  eye,  where  hypopyon  results.  The  most  interesting 
point,  however,  is  with  regard  to  its  action  on  mucous  surfaces ;  for,  while 
in  the  case  of  animals  it  produces  no  effect,  its  introduction  into  the  human 
urethra  causes  acute  catarrh,  attended  with  purulent  discharge.  He  found 
that  no  tolerance  to  the  toxin  resulted  after  five  successive  injections  at 
intervals.  In  a  recent  publication  he  points  out  that  the  toxin  has  marked 
effects  on  intracerebral  injection ;  he  also  claims  to  have  produced  an  anti- 
toxin. He  claims  that  the  toxin  diffuses  out  in  the  culture  medium,  and  does 
not  merely  result  from  disintegration  of  the  organisms.  This  has,  however, 
been  called  in  question  by  other  investigators. 

Distribution  in  the  Tissues.  —  The  gonococcus  having  been 
thus  shown  to  be  the  direct  cause  of  the  disease,  some  additional 
facts  may  be  given  regarding  its  presence  both  in  the  primary 
and  secondary  lesions.  In  the  human  urethra  the  gonococci 
penetrate  the  mucous  membrane,  passing  chiefly  between  the 
epithelial  cells,  causing  a  loosening  and  desquamation  of  many 
of  the  latter  and  inflammatory  reaction  in  the  tissues  below, 
attended  with  great  increase  of  secretion.  There  occurs  also 
a  gradually  increasing  emigration  of  leucocytes,  which  take  up  a 


228  GONORRHCEA,   SOFT   SORE,    SYPHILIS. 

large  number  of  the  organisms.  The  organisms  also  penetrate 
the  subjacent  connective  tissue,  and  are  especially  found  along 
with  extensive  leucocytic  emigration  around  the  lacunae.  Here 
also  many  are  contained  within  leucocytes.  Even,  however, 
when  the  gonococci  have  disappeared  from  the  urethra!  dis- 
charge, they  may  still  be  present  in  the  deeper  part  of  the 
mucous  membrane  of  the  urethra,  possibly  also  in  the  prostate, 
and  may  thus  be  capable  of  producing  infection.  The  prostatic 
secretion  may  sometimes  be  examined  by  making  pressure  on 
the  prostate  from  the  rectum  when  the  patient  has  almost  emptied 
his  bladder,  the  secretion  being  afterwards  discharged  along  with 
the  remaining  urine.  (Foulerton.)  In  acute  gonorrhoea  there 
is  often  a  considerable  degree  of  inflammatory  affection  of  the 
prostate  and  vesiculae  seminales,  but  whether  these  conditions 
are  always  due  to  the  presence  of  gonococci  in  the  affected 
parts  we  have  not  at  present  the  data  for  determining.  A  similar 
statement  also  applies  to  the  occurrence  of  orchitis  and  also  of 
cystitis  in  the  early  stage  of  gonorrhoea.  Gonococci  have,  how- 
ever, been  obtained  in  pure  culture  from  periurethral  abscess 
and  from  epididymitis.  During  the  more  chronic  stages  other 
organisms  may  appear  in  the  urethra,  aid  in  maintaining  the 
irritation,  and  produce  some  of  the  secondary  results.  The 
bacillus  coli,  the  pyogenic  cocci,  etc.,  are  often  present,  and 
may  extend  along  the  urethra  to  the  bladder  and  set  up  cystitis, 
though  in  this  they  may  be  aided  by  the  passage  of  a  catheter. 
It  is  then  also  that  buboes  usually  occur,  often  associated  with 
the  presence  of  a  small  ulcer  in  the  urethra.  Though  the 
bacteriology  of  these  cannot  yet  be  said  to  be  fully  worked  out, 
they  are  certainly  sometimes  produced  by  the  ordinary  pyogenic 
organisms  and  by  some  varieties  of  diplococci  which  are  often 
present  in  the  urethra  in  abnormal  conditions.  It  may  be 
mentioned  here  that  Wertheim  cultivated  the  gonococcus  from 
a  case  of  chronic  gonorrhoea  of  two  years'  standing,  and  by 
inoculation  on  the  human  subject  proved  it  to  be  still  virulent. 

In  the  disease  in  the  female,  gonococci  are  almost  invariably 
present  in  the  urethra,  the  situation  affected  next  in  frequency 
being  the  cervix  uteri.  They  do  not  appear  to  infect  the  lining 
epithelium  of  the  vagina  of  the  adult  unless  some  other  abnor- 
mal condition  be  present,  but  they  do  so  in  the  gonorrhceal 
vulvo-vaginitis  of  young  subjects.  They  have  also  been  found 


GONOCOCCUS    IN   JOINT   AFFECTIONS,   ETC.  229 

in  suppurations  in  connection  with  Bartholini's  glands,  and  some- 
times produce  an  inflammatory  condition  of  the  mucous  mem- 
brane of  the  body  of  the  uterus.  They  may  also  pass  along  the 
Fallopian  tubes  and  produce  inflammation  of  the  mucous  mem- 
brane there.  From  the  pus  in  cases  of  pyosalpinx  they  have 
been  cultivated  in  a  considerable  number  of  cases.  According 
to  the  results  of  various  observers  they  are  present  in  one  out 
of  four  or  five  cases  of  this  condition,  usually  unassociated  with 
other  organisms.  Further,  in  a  large  proportion  of  the  cases  in 
which  the  gonococcus  has  not  been  found  no  organisms  of  any 
kind  have  been  obtained  from  the  pus,  and  in  these  cases  the 
gonococci  may  have  been  once  present,  and  have  subsequently 
died  out.  Lastly,  they  may  pass  to  the  peritoneum  and  produce 
peritonitis,  which  is  usually  of  a  local  character,  but  cases  of 
acute  diffuse  peritonitis  are  recorded  by  Meija,  Frank,  Gushing, 
Runner  and  Harris.  It  is  chiefly  to  the  methods  of  culture 
supplied  by  Wertheim  that  we  owe  our  extended  knowledge  of 
such  conditions. 

In  gonorrhosal  conjunctivitis  the  mode  in  which  the  gonococci 
spread  through  the  epithelium  to  the  subjacent  connective 
tissue  is  closely  analogous  to  what  obtains  in  the  case  of  the 
urethra.  Their  relation  to  the  leucocytes  in  the  purulent  secre- 
tion is  also  the  same.  Microscopic  examination  of  the  secretion 
alone  in  acute  cases  often  gives  positive  evidence,  and  pure  cul- 
tures may  be  readily  obtained  on  blood  agar.  As  the  condition 
becomes  more  chronic  the  gonococci  are  less  numerous  and  a 
greater  portion  of  other  organisms  may  be  present. 

Relations  to  Joint  Affections,  etc.  —  The  relations  of  the  gono- 
coccus to  the  sequelae  of  gonorrhoea  form  a  subject  of  great 
interest  and  importance,  and  the  application  of  recent  methods 
of  examination  show  that  the  organism  is  much  more  frequently 
present  in  such  conditions  than  the  earlier  results  indicated. 
The  following  statements  may  be  made  with  regard  to  them. 
First,  in  a  considerable  number  of  cases  of  arthritis  following 
gonorrhoea,  the  gonococcus  has  been  found  microscopically, 
and  pure  cultures  have  been  obtained,  e.g.  by  Neisser,  Lang, 
Bordoni-Uffreduzzi,  and  many  others.  A  similar  statement 
applies  to  inflammation  of  the  sheaths  of  tendons  following 
gonorrhoea.  Secondly,  in  a  large  proportion  of  cases  no  organ- 
isms have  been  found.  It  is,  however,  possible  that  in  a  number 


230  GONORRHCEA,    SOFT   SORE,    SYPHILIS. 

of  these  the  gonococci  may  have  been  present  in  the  synovial 
membrane,  as  it  has  been  observed  that  they  may  be  much  more 
numerous  in  that  situation  than  in  the  fluid.  Thirdly,  in  some 
cases,  especially  in  those  associated  with  extensive  suppuration, 
occasionally  of  a  pyaemic  nature,  various  pyogenic  cocci  have 
been  found  to  be  present.  In  the  instances  in  which  the  gono- 
coccus  has  been  found  in  the  joints,  the  fluid  present  has  been 
described  as  being  usually  of  a  whitish-yellow  tint,  somewhat 
turbid,  and  containing  shreds  of  fibrin-like  material,  sometimes 
purulent  in  appearance.  In  one  case  Bordoni-Uffreduzzi  culti- 
vated the  gonococcus  from  a  joint  affection,  and  afterwards 
produced  gonorrhoea  in  the  human  subject  by  inoculating  with 
the  cultures  obtained.  In  another  case  in  which  pleurisy  was 
present  along  with  arthritis  the  gonococcus  was  cultivated  from 
the  fluid  in  the  pleural  cavity.  The  existence  of  a  gonorrJioeal 
endocarditis  has  been  established  by  recent  observations.  Cases 
apparently  of  this  nature  occurring  in  the  course  of  gonorrhoea 
had  been  previously  described,  but  the  complete  bacteriological 
test  has  now  been  satisfied  in  several  instances.  In  one  case 
Lenhartz  produced  gonorrhoea  in  the  human  subject  by  inocu- 
lation with  the  organisms  obtained  from  the  vegetations.  That 
a  true  gonorrhceal  septiccemia  may  also  occur  has  also  been 
established,  cultures  of  the  gonococcus  having  been  obtained 
from  the  blood  during  life  on  more  than  one  occasion  (Thayer 
and  Blumer,  Thayer  and  Lazear,  Ahmann,  Wilson,  and  Harris 
and  Johnston). 

Methods  of  Diagnosis.  —  For  microscopical  examination  dried 
films  of  the  suspected  pus,  etc.,  may  be  stained  by  any  of  the 
simple  solutions  of  the  basic  aniline  stains.  We  prefer  methy- 
lene,  or  thionin-blue,  as  they  do  not  overstain,  and  the  films  do 
not  need  to  be  decolorised.  Staining  for  one  minute  is  sufficient. 
It  is  also  advisable  to  stain  by  Gram's  method,  and  it  is  a  good 
plan  to  put  at  one  margin  of  the  cover-glass  a  small  quantity  of 
culture  of  staphylococcus  aureus  if  available,  in  order  to  have  a 
standard  by  which  to  be  certain  that  the  supposed  gonococci  are 
really  decolorised.  Regarding  the  value  of  microscopic  examina- 
tion alone,  we  may  say  that  the  presence  of  a  large  number  of 
micrococci  in  a  urethral  discharge  having  the  characters,  position, 
and  staining  reactions  described  above,  is  practically  conclusive 
that  the  case  is  one  of  gonorrhoea.  There  is  no  other  condition 


SOFT  SORE. 


231 


in  which  the  sum  total  of  the  microscopical  characters  is  present. 
We  consider  that  it  is  sufficient  for  purposes  of  clinical  diagnosis, 
and  therefore  of  great  value ;  in  the  acute  stage  a  diagnosis  can 
thus  be  made  earlier  than  by  any  other  method.  The  mistake  of 
confusing  gonorrhoea  with  such  conditions  as  a  urethral  chancre 
with  urethritis,  will  also  be  avoided.  Even  in  chronic  cases  the 
typical  picture  is  often  well  maintained,  and  microscopic  exam- 
ination alone  gives  a  definite  positive  result.  When  other  organ- 
isms are  present,  and  especially  when  the  gonococci  are  few  in 
number,  it  is  difficult,  and  in  some  cases  impossible,  to  give  a 
definite  opinion,  as  a  few  gonococci  mixed  with  other  organisms 
cannot  be  recognised  with  certainty.  This  is  often  the  condition 
in  chronic  gonorrhoea  in  the  female.  Microscopic  examination, 
therefore,  though  often  giving  positive  results,  will  sometimes 
be  inconclusive.  As  regards  lesions  in  other  parts  of  the  body, 
microscopic  examination  alone  is  quite  insufficient ;  it  is  practi- 
cally impossible,  for  example,  to  distinguish  by  this  means  the 
gonococcus  from  the  diplococcus  intracellularis  of  meningitis. 
Cultures  alone  supply  the  absolute  test,  and  this  test  should 
never  be  neglected  when  a  diagnosis  is  rendered  absolutely 
necessary  in  reference  to  moral  social  status,  or  to  medico-legal 
inquiry.  We  then  have  recourse  to  the  plate  method,  using 
Wertheim's  medium,  or  hydrocele-fluid  agar. 

SOFT  SORE. 

Within  recent  years  a  considerable  amount  of  attention  has 
been  directed  to  the  bacteriology  of  this  condition,  owing  to  the 
discovery  of  a  somewhat  characteristic  bacillus  in  the  affected 
parts.  This  organism  was  first  described  by  Ducrey  in  1889,  who 
found  it  in  the  purulent  discharge  from  the  ulcerated  surface ; 
and,  later,  in  1892,  Unna  described  its  appearance  and  distribution 
as  seen  in  sections  through  the  sores.  The  statements  of  these 
observers  regarding  the  presence  and  characters  of  this  organism 
have  been  fully  confirmed  by  other  observers. 

Microscopical  Characters,  — This  organism  appears  in  the  form 
of  minute  oval  rods  measuring  about  1.5  /^  in  length,  and  .5  /* 
in  thickness.  It  is  found  mixed  with  other  organisms  in  the 
purulent  discharge  from  the  surface,  and  is  chiefly  arranged  in 
small  groups  or  in  short  chains.  When  studied  in  sections 
through  the  ulcer  it  is  found  in  the  superficial  part  of  the  floor, 


232  GONORRHOEA,    SOFT    SORE,    SYPHILIS. 

but  more  deeply  situated  than  other  organisms,  and  may  be 
present  in  a  state  of  purity  amongst  the  leucocytic  infiltration. 
In  this  position  it  is  usually  arranged  in  chains  which  may  be 
of  considerable  length,  and  which  are  often  seen  lying  in  parallel 
rows  between  the  cells.  The  bacilli  chiefly  occur  in  the  free  con- 
dition, but  occasionally  a  few  may  be  contained  within  leucocytes. 

This  bacillus  takes  up  the  basic  aniline  stains  fairly  readily, 
but  loses  the  colour  very  rapidly  when  a  decolorising  agent  is 
applied.  Accordingly,  in  film  preparations  when  dehydration  is 
not  required,  it  can  be  readily  stained  by  most  of  the  ordinary 
combinations,  though  LofBer's  or  Kiihne's  methylene-blue  solu- 
tions are  preferable,  as  they  do  not  overstain.  In  sections,  how- 
ever, great  care  must  be  taken  in  the  process  of  dehydration, 
and  the  aniline-oil  method  (vide  p.  96)  should  be  used  for  this 
purpose,  as  alcohol  decolorises  the  organism  very  readily.  A 
little  of  the  methylene-blue  or  other  stain  may  be  with  advantage 
added  to  the  aniline  oil  used  for  dehydrating. 

This  organism  has  not  yet  been  successfully  cultivated  out- 
side the  body,  though  practically  every  medium  has  been  tried 
for  this  purpose.  Ducrey,  however,  succeeded  in  separating  it 
from  other  organisms  by  the  following  method.  He  produced 
a  series  of  pustules  by  successive  inoculations  in  the  human 
subject  on  the  skin,  which  had  been  previously  sterilised,  the 
pustules  being  afterwards  protected  from  contamination  by 
watch-glasses  fixed  in  position.  He  found  that  in  this  method 
the  other  organisms  gradually  died  off,  while  the  characteristic 
bacilli  persisted,  and  at  about  the  fifth  or  sixth  inoculation  might 
be  present  alone.  Further,  the  pus  containing  the  bacilli  in  a 
pure  condition  still  produced  the  typical  lesion  on  inoculation. 
Even  when  the  organisms  were  thus  separated  he  failed  to 
obtain  any  growth  on  the  numerous  media  which  he  employed. 

The  evidence  that  this  organism  is  the  causal  agent  in  the 
affection  accordingly  rests  on  the  facts  well  established  that  the 
organism  is  apparently  always  present  in  the  discharge  from 
the  sore,  and  in  its  tissues  ;  that  it  has  been  observed  hitherto  in 
no  other  form  of  ulceration  ;  and  that  it  is  sharply  marked  off 
from  saprophytic  organisms  by  the  fact  that  it  has  not  been 
obtained  in  cultures  outside  the  body. 

Regarding  the  presence  of  this  organism  in  the  buboes  asso- 
ciated with  soft  sore,  there  is  some  uncertainty. ,  A  considerable 


SOFT    SORE,  SYPHILIS.  233 

number  of  observers  have  failed  to  find  it,  and  have  also  failed 
to  produce  a  characteristic  soft  sore  by  inoculation  with  pus 
withdrawn  from  a  bubo  under  aseptic  precautions.  When  a 
chancroid  condition  follows  in  a  bubo  which  has  been  opened, 
they  accordingly  consider  that  it  has  been  secondarily  inocu- 
lated with  the  bacillus.  On  the  other  hand,  one  or  two  observ- 
ers have  found  the  bacillus  in  unopened  buboes.  Audry,  for 
example,  in  a  bubo  before  suppuration  had  occurred,  found  it 
lying  in  little  groups  of  two  or  three  within  leucocytes  in  the 
lymph  channels ;  and  in  this  case  inoculation  with  the  material 
from  the  bubo  produced  the  typical  lesion.  Krefting  also  found 
it  in  buboes  in  some  cases.  It  is  therefore  possible  that  the 
buboes  associated  with  soft  sore  are  caused  by  the  same  organ- 
isms, but  that  as  suppuration  occurs  they  in  great  part  die  off. 
It  seems  certain  at  least,  from  the  results  of  various  workers, 
that  in  many  cases  the  ordinary  pyogenic  organisms  are  not 
present  in  the  suppurating  buboes. 

In  connection  with  the  two  diseases,  gonorrhoea  and  soft 
sore,  it  is  of  special  interest  to  note  in  the  case  of  the  former 
how  restricted  are  the  conditions  of  growth  outside  the  body  of 
the  organism  which  produces  the  disease,  and  in  the  case  of  the 
latter,  that  attempts  to  cultivate  the  supposed  causal  organism 
outside  the  body  have  entirely  failed. 

However,  Besangon,  Griffon  and  Le  Sourd  claim  to  have 
grown  Ducrey's  bacillus  on  human  blood  agar,  as  well  as  on 
that  of  the  dog  and  the  hare,  where  all  the  morphological  peculi- 
arities before  described  were  reproduced.  In  the  condensation- 
water  growth  in  tube-cultures,  the  bacilli  grew  out  in  long  wavy 
chains,  whilst  in  uncoagulated  hare's  blood  the  bacilli  were  so 
short  as  to  resemble  chains  of  streptococci.  The  viability  and 
virulence  of  blood-agar  cultures  were  maintained  for  a  relatively 
long  period :  a  culture  of  the  eleventh  generation  still  produced 
typical  chancres,  although  on  hare's  blood  the  viability  was  very 
brief.  Due  credence  cannot  be  given  to  this  research  until 
further  confirmation. 

SYPHILIS. 

Regarding  the  relation  of  bacteria  to  this  disease,  we  cannot 
be  said  at  present  to  possess  much  definite  knowledge.  Most 
interest,  however,  is  attached  to  the  observations  of  Lustgarten, 


234  GONORRHCEA,    SOFT    SORE,    SYPHILIS. 

who  in  1884  described  a  characteristic  bacillus  both  in  the  pri- 
mary sore  and  in  the  lesions  in  internal  organs.  He  found  it 
in  all  of  sixteen  cases  which  he  examined.  This  bacillus  some- 
what resembles  the  tubercle  bacillus  in  shape  and  size.  It  occurs 
in  the  form  of  slender  rods,  straight  or  slightly  bent,  about  3  to 
4  fi  in  length,  often  forming  little  clusters  either  within  cells  or 
lying  free  in  the  lymphatic  spaces.  Like  the  tubercle  bacillus 
it  takes  up  the  basic  aniline  stains  with  difficulty,  but  it  is  much 
more  easily  decolorised  by  mineral  acids.  Lustgarten  stained 
the  tissues  for  twenty-four  to  forty-eight  hours  in  aniline-water 
solution  of  gentian-violet ;  and  then,  after  washing  them  in 
alcohol,  placed  them  for  ten  seconds  in  a  1.5  per  cent  solution 
of  permanganate  of  potassium.  They  were  then  treated  with 
sulphurous  acid,  which  removes  the  brown  precipitate  formed, 
and  decolorises  the  sections.  They  were  then  washed  in  water, 
dehydrated,  and  mounted.  The  observations  of  other  workers 
have  given  contradictory  results.  De  Michele  and  Radice,  for 
example,  found  Lustgarten's  bacilli  in  the  tissues  in  forty-five 
out  of  sixty-four  cases  examined,  while,  on  the  other  hand,  other 
observers  have  failed  to  find  them. 

Apart,  however,  from  negative  results  obtained  by  many, 
criticism  has  been  made  in  other  ways.  It"  has  been  alleged  by 
some  that  Lustgarten's  bacilli  is  merely  the  smegma  bacillus  which 
has  penetrated  the  affected  tissues.  This  explanation,  however, 
would  not  account  for  the  presence  of  the  bacilli  in  the  internal 
organs,  where  they  were  observed  by  Lustgarten  and  others, 
And  further,  there  are  minor  points  of  difference  between  this 
smegma  bacillus  and  Lustgarten's  bacillus.  It  has  also  been 
suggested  by  some  that  the  organisms  described  by  Lustgarten 
are  merely  tubercle  bacilli  which  have  been  accidentally  present 
in  the  affected  tissues.  Those,  however,  who  have  found  the 
former  organism  in  the  tissues  agree  that  it  can  be  readily 
distinguished  from  the  tubercle  bacillus,  as  it  does  not  resist 
decolorising  with  strong  acids.  This  explanation  of  the  presence 
of  these  bacilli  in  the  tissues  is  really  without  definite  support. 

The  organism  has  not  been  cultivated  outside  the  body, 
though,  in  view  of  what  we  know  with  regard  to  some  other 
diseases,  this  fact  in  itself  does  not  form  a  grave  objection.  In 
the  absence,  however,  of  definite  evidence  as  to  its  invariable 
presence  in  the  lesions,  its  relations  to  the  disease  are  still  highly 


SYPHILIS. 


235 


problematical.  It  may  also  be  noticed  that  this  organism  has 
been  found  in  the  tertiary  lesions,  which  are  usually  believed  to 
be  non-infectious. 

Other  organisms  have  been  described  as  present  in  syphilitic 
lesions,  notably  one  quite  recently  by  Van  Niessen.  This 
organism  is  a  pleomorphous  bacillus  belonging  to  the  higher 
bacteria.  He  claims  not  only  to  have  demonstrated  it,  both  in 
the  tissues  and  in  the  blood,  but  to  have  obtained  it  in  pure 
culture  from  a  number  of  cases.  On  the  other  hand,  Schiiller 
states  that  he  has  found  a  protozoon-like  organism  in  a  great 
variety  of  syphilitic  lesions.  The  latest  researches  are  those 
reported  by  Joseph  and  Piorkowski,  who  have  cultivated  on 
sterile  placentae  a  certain  bacillus  from  the  semen  of  twenty-five 
syphilitics  and  from  the  blood  in  two  cases.  Their  description 
of  the  bacillus  shows  it  to  have  many  characters  in  common 
with  those  organisms  which  resemble  B.  diphtherias.  Four 
-cases  supposed  to  be  free  of  syphilitic  taint  when  examined 
in  the  same  manner  showed  none  of  the  bacilli.  Until  con- 
firmation of  these  results  has  been  obtained  it  is  unnecessary  to 
give  details. 


CHAPTER  X. 

TUBERCULOSIS. 

THE  cause  of  tubercle  was  proved  by  Koch  in  1882  to  be  the 
organism  now  universally  known  as  the  tubercle  bacillus.  Prob- 
ably no  other  single  discovery  has  had  a  more  important  effect 
on  medical  science  and  pathology  than  this.  It  has  not  only 
shown  what  is  the  real  cause  of  the  disease,  but  has  also  sup- 
plied infallible  methods  for  determining  what  are  tubercular 
lesions  and  what  are  not,  and  has  also  given  the  means  of  study- 
ing the  modes  and  paths  of  infection.  A  definite  answer  has  in 
this  way  been  supplied  to  many  questions  which  were  previously 
the  subject  of  endless  discussion. 

Historical.  —  Klencke  in  1843  made  the  statement  that  he  had  produced 
tuberculosis  in  rabbits  by  intravenous  injection  of  tubercular  material,  but  he 
only  concluded  from  these  experiments  that  the  cells  of  tubercles  could  multi- 
ply and  reproduce  the  disease,  and  he  appears  to  have  placed  little  importance 
on  the  discovery.  Villemin  has  the  honour  of  having  been  the  first  to  inves- 
tigate the  infectious  character  of  tubercle  by  systematic  experiments,  and  to 
demonstrate  the  regularity  with  which  tuberculosis  can  be  transmitted  by  in- 
oculation with  tubercular  material.  His  first  observations  were  published  in 
1865.  He  produced  tuberculosis  in  animals  not  only  by  tubercular  material 
from  the  human  subject,  but  also  by  portions  of  what  were  known  as  the  Perl- 
sucht  nodules  in  cattle,  and  came  to  the  conclusion  that  Perlsucht  was  due  to 
the 'same  virus  as  tubercle.  He  concluded  that  this  virus  was  comparable  in 
its  mode  of  action  with  that  of  other  infectious  diseases.  These  views,  how- 
ever, aroused  a  storm  of  opposition  from  all  sides.  The  opposition  was  at 
first  chiefly  on  theoretical  grounds,  but  later  also  from  experimental  results. 
Investigators  who  repeated  Villemin's  experiments  obtained  similar  results  so 
far  as  the  production  of  tuberculosis  by  tubercular  material  was  concerned, 
but  many  found  that  tuberculosis  also  followed  inoculation  with  non-tubercular 
material  (such  as  pus  from  pyaemic  abscesses,  portions  of  decomposed  tissue, 
etc.),  and  even  by  the  mere  introduction  of  setons.  The  general  opinion 
came  to  be  strongly  against  the  existence  in  tubercle  of  an  infective  agent 
of  specific  nature,  and  along  with  this  there  prevailed  great  confusion  as  to 
the  distinction  between  tubercular  and  non-tubercular  lesions. 

By  the  work  of  Armanni  and  of  Cohnheim  and  Salomonsen  (1870-80)  it 
had  been  demonstrated  that  tubercle  was  an  infective  disease.  The  latter 

236 


TUBERCULOSIS    IN   ANIMALS.  237 

observers  found  on  inoculation  of  the  anterior  chamber  of  the  eye  of  rabbits 
with  tubercular  material  that  in  many  cases  the  results  of  irritation  soon  dis- 
appeared, but  that  after  a  period  of  incubation,  usually  about  twenty-five  days, 
small  tubercular  nodules  appeared  in  the  iris  ;  afterwards  the  disease  gradually 
spread,  leading  to  a  tubercular  disorganisation  of  the  globe  of  the  eye.  Later, 
the  lymphatic  glands  became  involved,  and  finally  the  animal  died  of  acute 
tuberculosis.  The  question  remained  as  to  the  nature  of  the  virus,  the  spe- 
cific character  of  which  was  thus  established,  and  this  question  was  answered 
by  the  work  of  Koch. 

The  announcement  of  the  discovery  of  the  tubercle  bacillus  was  made  by 
Koch  in  March,  1882,  and  a  full  account  of  his  researches  appeared  in  1884 
(Mitth.  a.  d.  K.  Gsndhtsamte.,  Berlin).  Koch's  work  on  this  subject  will  re- 
main as  a  classical  masterpiece  of  bacteriological  research,  both  on  account  of 
the  great  difficulties  which  he  successfully  overcame  and  the  completeness 
with  which  he  demonstrated  the  relations  of  the  organism  to  the  disease.  The 
two  chief  difficulties  were,  first,  the  demonstration  of  the  bacilli  in  the  tissues, 
and,  secondly,  the  cultivation  of  the  organism  outside  the  body.  For,  with 
regard  to  the  first,  the  tubercle  bacillus  cannot  be  demonstrated  by  a  simple 
watery  solution  of  a  basic  aniline  dye,  and  it  was  only  after  prolonged  staining 
for  twenty-four  hours  with  a  solution  of  methylene-blue  with  caustic  potash 
added,  that  he  was  able  to  reveal  the  presence  of  the  organism.  Then,  in  the 
second  place,  all  attempts  to  cultivate  it  on  the  ordinary  media  failed,  and  he 
only  succeeded  in  obtaining  growth  on  solidified  blood  serum,  the  method  of 
preparing  which  he  himself  devised,  inoculations  being  made  on  this  medium 
from  the  organs  of  animals  artificially  rendered  tubercular.  The  fact  that 
growth  did  not  appear  till  the  tenth  day  at  the  earliest,  might  easily  have  led 
to  the  hasty  conclusion  that  no  growth  took  place.  All  difficulties  were,  how- 
ever, successfully  overcome.  He  cultivated  the  organism  by  the  above  method 
from  a  great  variety  of  sources,  and  by  a  large  series  of  inoculation  experiments 
on  various  animals,  performed  by  different  methods,  he  conclusively  proved 
that  bacilli  from  these  different  sources  produced  the  same  tubercular  lesions 
and  were  really  of  the  same  species.  His  work  was  the  means  of  showing 
conclusively  that  such  conditions  as  lupus,  "white  swelling"  of  joints,  scrofu- 
lous disease  of  glands,  etc.,  are  really  tubercular  in  nature. 

Tuberculosis  in  Animals. — Tuberculosis  is  not  only  the  most 
widely  spread  of  all  diseases  affecting  the  human  subject,  and 
produces  a  mortality  greater  than  any  other,  but  there  is  proba- 
bly no  other  disease  which  affects  the  domestic  animals  so  widely. 
We  need  not  here  describe  in  detail  the  various  tubercular  lesions 
in  the  human  subject,  but  some  facts  regarding  the  disease  in 
the  lower  animals  may  be  given,  as  this  subject  is  of  great  im- 
portance in  relation  to  the  infection  of  the  human  subject. 

Amongst  the  domestic  animals  the  disease  is  commonest  in  cattle  (bovine 
tuberculosis),  and  in  them  the  lesions  are  very  various,  both  in  their  character 


238  TUBERCULOSIS. 

and  distribution.  In  most  cases  the  lungs  are  affected,  and  contain  numerous, 
rounded  nodules,  many  being  of  considerable  size ;  these  may  be  softened  in 
the  centre,  but  are  usually  of  pretty  firm  consistence  and  may  be  calcified. 
There  may  be  in  addition  caseous  pneumonia,  and  also  small  tubercular  gran- 
ulations. Along  with  these  changes  in  the  lungs,  the  pleurae  are  also  often 
affected,  and  show  numerous  nodules,  some  of  which  may  be  of  large  size, 
firm  and  pedunculated,  the  condition  being  known  in  Germany  as  Perlsucht* 
in  France  as  poinvieltire.  Lesions  similar  to  the  last  may  be  chiefly  confined 
to  the  peritoneum  and  pleurae.  In  other  cases,  again,  the  abdominal  organs 
are  principally  involved.  The  udder  becomes  affected  in  a  certain  proportion 
of  cases  of  tuberculosis  in  cows  —  in  3  per  cent  according  to  Bang — but 
primary  affection  of  this  gland  is  very  rare.  Tuberculosis  is  also  a  compara- 
tively common  disease  in  pigs,  in  which  animals  it  in  many  cases  affects  the 
abdominal  organs,  in  other  cases  produces  a  sort  of  caseous  pneumonia,  and 
sometimes  is  met  with  as  a  chronic  disease  of  the  lymphatic  glands,  the  so- 
called  "  scrofula"  of  pigs.  Tubercular  lesions  in  the  muscles  are  less  rare  in 
pigs  than  in  most  other  animals.  In  the  horse  the  abdominal  organs  are  usu- 
ally the  primary  seat  of  the  disease,  the  spleen  being  often  enormously  enlarged 
and  crowded  with  nodules  of  various  shapes  and  sizes ;  sometimes,  however, 
the  primary  lesions  are  pulmonary.  In  sheep  and  goats  tuberculosis  is  a  rare 
occurrence,  especially  in  the  former  animals.  It  also  occurs  spontaneously  in 
dogs,  cats,  and  in  the  large  carnivora.  It  is  also  sometimes  met  with  in  mon- 
keys in  confinement,  and  leads  to  a  very  rapid  and  widespread  affection  in  these 
animals,  the  nodules  having  a  special  tendency  to  soften  and  break  down  into 
a  pus-like  fluid. 

Tuberculosis  in  fowls  (avian  tuberculosis)  is  a  common  and  very  infectious 
disease,  nearly  all  the  birds  in  the  poultry-yard  being  sometimes  affected. 
The  relation  of  the  different  forms  of  tuberculosis  is  discussed  below. 

From  these  statements  it  will  be  seen  that  the  disease  in  ani- 
mals presents  great  variations  in  character,  and  may  differ  in 
many  respects  from  that  met  with  in  the  human  subject.  The 
tubercle  nodules  may  be  of  so  large  a  size,  e.g.  in  the  horse  and 
ox,  as  to  be  described  as  sarcoma-like ;  they  may  be  tough  and 
firm,  with  little  or  no  caseation,  or  they  may  be  softened  in  the 
centre,  more  resembling  abscesses,  or  again  there  may  be  an 
eruption  of  very  minute  granulations.  However  different  their 
naked-eye  appearances  may  be,  they  are  built  up  histologically 
on  the  same  plan,  and  of  greater  importance  still  is  the  fact 
that  they  are  all  produced  by  the  tubercle  bacillus.  An 
account  of  the  lesions  experimentally  produced  will  be  given 
later. 

Tubercle  Bacillus.  —  Microscopical  Characters.  —  Tubercle 
bacilli  are  minute  rods  which  usually  measure  2.5  to  3.5  p  in 
length,  and  .3  ft  in  thickness,  i.e.  in  proportion,  to  their  length 


MORPHOLOGY   OF  TUBERCLE   BACILLUS. 


239 


they  are  comparatively  thin  organisms  (Figs.  87  and  88). 
Sometimes,  however,  longer  forms,  up  to  5  /*  or  more  in  length, 
are  met  with,  both  in  cultures 
and  in  the  tissues.  They  are 
straight  or  slightly  curved,  and 
are  of  uniform  thickness,  or  may 
show  slight  swelling  at  their 
extremities.  When  stained  they 
appear  uniformly  coloured,  or 
may  present  small  uncoloured 
spots  along  their  course,  with 
darkly  stained  parts  between. 
In  the  case  of  the  tubercle  ba- 
cillus, as  of  many  other  organ- 
isms, a  Considerable  amount  Of  FlG.  87.-Tubercle  bacilli,  from  a  pure 

discussion  has  taken  place  as  culture  on  glycerin  agar. 

, ,  ,.  Stained  with  carbol-fuchsin.     x  1000. 

to   the   occurrence    of    spores. 

In  such  a  minute  organism  it  is  extremely  difficult  to  recog- 
nise the  exact  characters  of  the  unstained  points.     Accordingly, 

we     find     that 

-A  some    consider 

these  to  be 
spores,  while 
others  find  that 
it  is  impossible 
to  stain  them 
by  any  means 
whatever,  and 
consider  that 
they  are  really 
of  the  nature 
of  vacuoles. 
Against  their 
being  spores  is 
also  the  fact 
that  many  oc- 
cur in  one  ba- 
Others 

again  hold  that 
some  of  the  condensed  and  highly  stained  particles  are  spores. 


FlG.  88.  —  Tubercle  bacilli  in  phthisical  sputum  ;  they  are  longer 
than  is  often  the  case.    Film  preparation,  stained  with  carbol-fuchsin    CllmS. 
and  methylene-blue.     X  1000. 


240  TUBERCULOSIS. 

It  is  impossible  to  speak  definitely  on  the  question  at  present. 
We  can  only  say  that  the  younger  bacilli  stain  uniformly,  and 
that  in  the  older  forms  inequality  in  staining  is  met  with,  but  it 
has  not  been  proved  that  this  indicates  spore  formation. 

The  bacilli  in  the  tissues  occur  scattered  irregularly  or  in 
little  masses.  They  are  usually  single,  or  two  are  attached  end 
to  end  and  often  form  in  such  a  case  an  obtuse  angle.  True 
chains  are  not  formed,  but  occasionally  short  filaments  are 
met  with.  In  cultures  the  bacilli  form  masses  in  which  the 
rods  are  closely  applied  to  one  another  and  arranged  in  a 
more  or  less  parallel  manner.  Tubercle  bacilli  are  quite  devoid 
of  motility. 

Aberrant  Forms.  —  Though  such  are  the  characters  of  the 
organism  as  usually  met  with,  other  appearances  are  sometimes 
found.  In  old  cultures,  for  example,  very  much  larger  elements 
may  occur.  These  may  be  in  the  form  of  long  filaments,  which 
may  be  swollen  or  clubbed  at  their  extremities,  may  be  irregularly 
beaded,  and  may  even  show  the  appearance  of  branching.  Such 
forms  have  been  studied  by  Metchnikoff,  Maffucci,  Klein,  and 
others.  Their  significance  has  been  variously  interpreted,  for 
while  some  look  upon  them  as  degenerated  or  involution  forms, 
others  regard  them  as  indicating  a  special  phase  in  the  life  history 
of  the  organism,  allying  it  with  the  higher  bacteria.  Recent 
observations,  however,  go  to  establish  the  latter  view,  and  this 
is  now  generally  accepted  by  authorities.  It  has  also  been 
found  that  under  certain  circumstances  tubercle  bacilli  in  the 
tissues  produce  a  radiating  structure  closely  similar  to  that 
of  the  actinomyces.  This  was  found  to  be  the  case  by  Babes 
and  also  by  Lubarsch,  when  the  bacilli  were  injected  under 
the  dura  mater  and  directly  into  certain  solid  organs,  such  as 
the  kidneys  in  the  -rabbit.  Club-like  structures  are  also  present 
at  the  periphery ;  these  are  usually  not  acid-fast,  but  they 
retain  the  stain  in  the  Weigert-Gram  method.  Similar  results 
have  also  been  obtained  with  other  acid-fast  bacilli,  which 
will  be  mentioned  below,  and  these  would  appear  to  form  a 
group  of  organisms  closely  allied  to  the  streptothriceae,  the 
bacillary  parasitic  form  being  one  stage  of  the  life  history  of 
the  organism. 

Staining  Reaction.  —  The  tubercle  bacillus  takes  up  the 
ordinary  stains  very  slowly  and  faintly,  and  for  successful  staining 


CULTIVATION    OF    TUBERCLE   BACILLUS.  241 

one  of  the  most  powerful  solutions  ought  to  be  employed,  e.g. 
gentian-violet  or  fuchsin,  along  with  aniline-oil  water  or  solution 
of  carbolic  acid.  Further,  such  staining  solutions  require  to  be 
applied  for  a  long  time,  or  the  staining  must  be  accelerated  by 
heat,  the  solution  being  warmed  till  steam  arises  and  the 
specimen  allowed  to  remain  in  the  hot  stain  for  two  or  three 
minutes.  One  of  the  best  and  most  convenient  methods  is  the 
Ziehl-Neelsen  method  (see  p.  104).  The  bacilli  present  this 
further  peculiarity,  however,  that  after  staining  has  taken  place 
they  resist  decolorising  by  solutions  which  readily  remove  the 
colour  from  the  tissues  and  from  other  organisms  which  may 
be  present.  Such  decolorising  agents  are  sulphuric  or  nitric 
acid  in  20  per  cent  solution,  or  2  per  cent  solution  of  hydro- 
chloric acid  in  80  per  cent  alcohol.  Preparations  can  thus  be 
obtained  in  which  the  tubercle  bacilli  alone  are  coloured  by  the 
stain  first  used,  and  the  tissues  can  then  be  coloured  by  a  con- 
trast stain.  Within  recent  years  certain  other  bacilli  have  been 
discovered  which  present  the  same  staining  reactions  as  tubercle 
bacilli;  they  are  therefore  called  " acid-fast  "  (vide  infra).  Tu- 
bercle bacilli,  also  stain  by  Gram's  method,  but  the  results  are 
inferior  to  those  obtained  with  carbolic  fuchsin. 

Bulloch  and  Macleod,  by  treating  tubercle  bacilli  with  hot  alcohol  and 
ether,  extracted  a  wax  which  gave  the  characteristic  staining  reactions  of  the 
bacilli  themselves.  The  remains  of  the  bacilli,  further,  when  extracted  with 
caustic  potash,  yielded  a  body  which  was  probably  a  chitin,  and  which  was 
acid-fast  when  stained  for  twenty-four  hours  with  carbol-fuchsin. 

Cultivation.  —  The  medium  first  used  by  Koch  was  inspissated 
blood  serum  (vide  p.  43).  If  inoculations  are  made  on  this 
medium  with  tubercular  material  free  from  other  organisms, 
there  appear  from  the  tenth  to  fourteenth  day  minute  points  of 
growth  of  dull  whitish  colour,  rather  irregular,  and  slightly  raised 
above  the  surface.  Koch  compared  the  appearance  of  these  to 
that  of  small  dry  scales.  In  such  cultures  they  usually  reach 
only  a  comparatively  small  size  and  remain  separate,  becoming 
confluent  only  when  many  occur  close  together.  In  sub-cultures, 
however,  growth  is  more  luxuriant  and  may  come  to  form  a  dull 
wrinkled  film  of  whitish  colour,  which  may  cover  the  greater 
part  of  the  surface  of  the  serum  and  at  the  bottom  of  the  tube 
may  grow  over  the  surface  of  the  condensation  water  on  to  the 


242 


TUBERCULOSIS. 


glass  (Fig.  89,  A).  The  growth  is  always  of  a  dull  appearance 
and  has  a  considerable  degree  of  consistence,  it  being  difficult 
to  dissociate  a  portion  thoroughly  in  a  drop  of  water.  In  older 
cultures  the  growth  may  acquire  a  slightly  brownish  or  buff 

colour.  When  the  small  colonies 
are  examined  under  a  low  power 
of  the  microscope  they  are  seen 
to  be  extending  at  the  periphery 
in  the  form  of  wavy  or  sinuous 
streaks  which  radiate  outward 
and  which  have  been  compared 
to  the  flourishes  of  a  pen.  The 
central  part  shows  similar  mark- 
ings closely  interwoven.  These 
streaks  are  composed  of  masses 
of  the  bacilli  arranged  in  a  more 
or  less  parallel  manner. 

On  glycerin  cigar,  which  was 
first  introduced  by  Nocard  and 
Roux  as  a  medium  for  the  culture 
of  the  tubercle  bacillus,  growth 
takes  place  in  sub-cultures  at 
an  earlier  date  and  progresses 
more  rapidly  than  on  serum, 
but,  strangely  enough,  this  me- 
dium is  not  suitable  for  obtaining 
cultures  from  the  tissues,  inocu- 
lations with  tubercular  material  usually  yielding  a  negative  re- 
sult. The  growth  has  practically  the  same  characters  as  on 
serum,  but  is  more  luxuriant.  The  organism,  however,  tends 
to  lose  its  virulence  more  rapidly  than  when  grown  on  serum. 
In  glycerin  broth,  especially  when  the  layer  is  not  deep,  tubercle 
bacilli  grow  readily  in  the  form  of  little  white  masses  which 
fall  to  the  bottom  and  form  a  powdery  layer.  If,  however,  the 
growth  be  started  on  the  surface  it  spreads  superficially  as  a 
dull  whitish,  wrinkled  pellicle  which  may  reach  the  walls  of  the 
flask ;  this  mode  of  growth  is  specially  suitable  for  the  produc- 
tion of  tuberculin  (vide  infra).  The  culture  has  a  peculiar 
fruity  and  not  unpleasant  odour.  On  ordinary  agar  and  on 
gelatin  media  no  growth  takes  place. 


ABC 

FIG.  89.  —  Cultures  of  tubercle  bacilli  on 

glycerin  agar. 

A  and  B.  Mammalian  tubercle  bacilli;  A  is 
an  old  culture,  B,  one  of  a  few  weeks'  growth. 
C.  Avian  tubercle  bacilli.  The  growth  is 
whiter  and  smoother  on  the  surface  than  the 
others. 


POWERS   OF   RESISTANCE. 


243 


It  was  at  one  time  believed  that  the  tubercle  bacillus  would  only  grow  on 
media  containing  animal  fluids,  but  of  late  years  it  has  been  found  that  growth 
takes  place  also  on  a  purely  vegetable  medium,  as  was  first  shown  by  Pawlowsky 
in  the  case  of  potatoes.  Sander  has  shown  that  the  bacillus  grows  readily  on 
potato,  carrot,  macaroni,  and  on  infusion  of  these  substances,  especially  when 
glycerin  is  added.  He  also  found  that  cultures  from  tubercular  lesions  could 
be  obtained  on  glycerin  potato  (p.  47).  In  sub-culture  the  bacillus  also 
grows  well  upon  potato  which  has  been  sterilised  in  2  per  cent  glucose  broth 
after  the  manner  of  glycerinated  potato. 

The  optimum  temperature  for  growth  is  37°  to  38°  C. 
Growth  ceases  above  42°  and  usually  below  28°,  but  on  long- 
continued  cultivation  outside  the  body  and  in  special  circum- 
stances, growth  may  take  place  at  a  lower  temperature,  e.g. 
Sander  found  that  growth  took  place  in  glycerin-potato  broth 
even  at  22°  to  23°  C. 

Powers  of  Resistance.  —  Tubercle  bacilli  have  considerable 
powers  of  resistance  to  external  influences,  and  can  retain  their 
vitality  for  a  long  time  outside  the  body  in  various  conditions ; 
in  fact,  in  this  respect  they  may  be  said  to  occupy  an  inter- 
mediate position  between  spores  and  spore-free  bacilli.  Dried 
phthisical  sputum  has  been  found  to  contain  still  virulent  bacilli 
(or  their  spores?)  after  two  months,  and  similar  results  .are 
obtained  when  the  bacilli  are  kept  in  distilled  water  for  several 
weeks.  So  also  they  resist  for  a  long  time  the  action  of  putre- 
faction, which  is  rapidly  fatal  to  many  pathogenic  organisms. 
Sputum  has  been  found  to  contain  living  tubercle  bacilli  even 
after  being  allowed  to  putrefy  for  several  weeks  (Fraenkel, 
Baumgarten),  and  the  bacilli  have  been  found  to  be  alive  in 
tubercular  organs  which  have  been  buried  in  the  ground  for  a 
similar  period.  They  are  not  killed  by  being  exposed  to  the 
action  of  the  gastric  juice  for  six  hours,  or  to  a  temperature  of 
-  3°  C.  for  three  hours,  even  when  this  is  repeated  several  times. 
It  has  been  found  that  when  completely  dried  they  can  resist  a 
temperature  of  100°  C.  for  an  hour,  but,  on  the  other  hand, 
exposure  in  the  moist  condition  to  70°  C.  for  the  same  time  is 
usually  fatal.  Theobald  Smith,  from  an  interesting  series  of 
thermal  death-point  tests,  concludes  that  bacilli  suspended  in 
distilled  water,  normal  salt  solution,  bouillon  and  milk  are 
destroyed  at  60°  C.  in  15  to  20  minutes,  the  larger  number 
being  destroyed  in  5  to  10  minutes.  In  milk  suspensions,  how- 
ever, the  pellicle  which  forms  during  heating  at  60°  C.  may 


244  TUBERCULOSIS. 

contain  living  bacilli  after  one  hour.  It  may  be  stated  that 
raising  the  temperature  to  100°  C.  kills  the  bacilli  in  fluids  and 
in  tissues,  but  in  the  case  of  large  masses  of  tissue  care  must  be 
taken  that  this  temperature  is  reached  throughout.  They  are 
killed  in  less  than  a  minute  by  exposure  to  5  per  cent  carbolic 
acid,  and  both  Koch  and  Straus  found  that  they  are  rapidly 
killed  by  being  exposed  to  the  action  of  direct  sunlight. 

Action  on  the  Tissues.  —  The  local  lesion  produced  by  the 
tubercle  bacillus  is  the  well-known  tubercle  nodule,  but  though 
the  typical  structure  is  often  described  as  consisting  of  a  central 
giant-cell  surrounded  by  a  zone  of  comparatively  large  and 
somewhat  spindle-shaped  cells  (epithelioid  cells),  and  again  by 
an  outer  zone  of  lymphocytes  or  small  uninucleated  leucocytes, 
the  structure  varies  in  different  situations  and  according  to  the 
intensity  of  the  action  of  the  bacilli. 

A  considerable  discussion  has  taken  place  as  to  the  exact 
origin  of  the  elements  composing  the  tubercle  follicle.  In  the 
case  of  the  iris  its  formation  was  fully  studied  by  Baumgarten, 
and  his  views  we  consider  to  be  correct  regarding  the  ordinary 
mode  of  formation.  Before  describing  the  exact  changes  which 
occur  in  the  tissues,  it  may  be  stated  that  the  action  of  the 
bacillus  is  twofold.  On  the  one  hand  it  induces  tissue  reaction 
in  the  form  of  leucocytic  infiltration  and  proliferative  changes, 
and  on  the  other  hand,  it  causes  degenerative  changes  in  the 
cells  around,  which  afterwards  result  in  their  death. 

After  the  bacilli  gain  entrance  to  a  connective  tissue  such  as 
that  of  the  iris,  their  first  action  appears  to  be  on  the  connective- 
tissue  cells,  which  become  somewhat  swollen  and  undergo  mitotic 
division,  the  resulting  cells  being  distinguishable  by  their  large 
size  and  pale  nuclei.  These  constitute  the  so-called  epithelioid 
cells.  These  proliferative  changes  may  be  well  seen  on  the  fifth 
day  after  inoculation  or  even  earlier.  A  small  focus  of  prolifer- 
ated cells  is  thus  formed  in  the  neighbourhood  of  the  bacilli 
and  about  the  same  time  numbers  of  leucocytes  —  chiefly  lym- 
phocytes—  begin  to  appear  at  the  periphery  and  gradually 
become  more  numerous. 

Soon,  however,  the  action  of  the  bacilli  as  cell-poisons  comes 
into  prominence,  the  changes  first  occurring  in  the  centre  of  the 
focus.  The  epithelioid  cells  become  swollen  and  somewhat 
hyaline,  their  outlines  become  indistinct,  whilst  their  nucleus 


ACTION   ON   THE   TISSUES.  245 

stains  Vaintly,  and  ultimately  loses  the  power  of  staining.  The 
cells  in\the  centre,  thus  altered,  gradually  become  fused  into  a 
homogeneous  substance  and  this  afterwards  becomes  somewhat 
granular  in  appearance.  If  the  central  necrosis  does  not  take 
place  quickly,  then  giant-cell  formation  may  occur  in  the  centre 
of  the  follicle,  this  constituting  one  of  the  characteristic  features, 
of  the  tubercular  lesion,  or  after  the  occurrence  of  caseation: 
giant-cells  may  be  formed  in  the  cellular  tissue  around.  The- 
centre  of  a  giant-cell  often  shows  signs  of  degeneration,  s-uch 
as  hyaline  change  and  vacuolation,  or  it  may  be  more  granular 
than  the  rest  of  the  cell. 

Though  there  has  been  a  considerable  amount  of  discussion 
as  to  the  mode  of  origin  of  the  giant-cells,  we  think  there  can 
be  little  doubt  that  in  most  cases  they  result  from  enlargement 
of  single  epithelioid  cells,  the  nucleus  of  which  undergoes  pro- 
liferation without  the  protoplasm  dividing.  These  epithelioid 
cells  may  sometimes  be  the  lining  cells  of  capillaries.  Sometimes 
cells  a  little  larger  than  epithelioid  cells  may  be  seen,  which 
contain  only  two  or  three  nuclei ;  these  may  be  young  giant-cells. 
Some  consider  that  the  giant-cells  result  from  a  fusion  of  the 
epithelioid  cells  ;  but,  though  there  are  occasionally  appearances 
which  indicate  such  a  mode  of  formation,  it  cannot  be  regarded 
as  of  common  occurrence.  In  some  cases  of  acute  tuberculosis, 
when  the  bacilli  become  lodged  in  a  capillary  the  endothelial 
cells  of  its  wall  may  proliferate,  and  thus  a  ring  of  nuclei  be 
formed  round  a  small  central  thrombus.  Such  an  occurrence 
gives  rise  to  an  appearance  closely  resembling  a  typical  giant- 
cell.  According  to  the  view  here  stated,  both  the  epithelioid  and 
the  giant-cells  are  of  connective  tissue  origin  ;  and  we  can  see  no 
sufficient  evidence  for  the  view  held  by  some  observers,  chiefly 
of  the  French  school,  that  they  are  formed  from  leucocytes 
which  have  emigrated  from  the  capillaries. 

There  can  be  no  doubt,  we  think,  from  a  careful  study  of  the 
tubercular  lesions,  that  the  cell  necrosis  and  subsequent  caseation 
depend  upon  the  products  of  the  bacilli,  and  are  not  due  to 
the  fact  that  the  tubercle  nodule  is  non-vascular.  This  non- 
vascularity  itself  is  to  be  explained  by  the  circumstance  that 
young  capillaries  cannot  grow  into  a  part  where  tubercle  bacilli 
are  active,  and  that  the  already  existing  capillaries  become 
thrombosed,  owing  to  the  action  of  the  bacillary  products  on 


246  TUBERCULOSIS. 

their  walls,  and  ultimately  disappear.  At  the  periphery  of 
tubercular  lesions  there  may  be  considerable  vascularity  and 
new  formation  of  capillaries. 

The  general  symptoms  of  tuberculosis  —  pyrexia,  perspiration, 
wasting,  etc.,  are  to  be  ascribed  to  the  absorption  and  distribu- 
tion throughout  the  system  of  the  toxic  products  of  the  bacilli ; 
in  the  case  of  phthisical  cavities  and  like  conditions  where  other 
bacteria  are  present,  the  toxins  of  the  latter  also  play  an  im- 
portant part.  The  occurrence  of  waxy  change  in  the  organs  is 
believed  by  some  to  be  chiefly  due  to  the  products  of  other, 
especially  pyogenic,  organisms,  secondarily  present  in  the  tuber- 
cular lesions.  This  matter,  however,  requires  further  elucidation. 

Presence  and  Distribution  of  tJie  Bacilli.  —  A  few  facts  may 
be  stated  regarding  the  presence  of  bacilli,  and  the  numbers  in 
which  they  are  likely  to  be  found  in  tubercular  lesions.  On  the 
one  hand,  they  may  be  very  few  in  number  and  difficult  to  find, 
and  on  the  other  hand,  they  may  be  present  in  very  large  num- 
bers, sometimes  forming  masses  which  are  easily  visible  under 
the  low  power  of  the  microscope. 

They  are  usually  very  few  in  number  in  chronic  lesions, 
whether  these  are  tubercle  nodules  with  much  connective  tissue 
formation  or  old  caseous  collections.  In  caseous  material  one 
can  sometimes  see  a  few  bacilli  faintly  stained,  along  with  very 
minute  unequally  stained  granular  points,  some  of  which  may 
possibly  be  spores  of  the  bacilli.  Whether  they  are  spores  or 
not,  the  important  fact  has  been  established  that  tubercular 
material  in  which  no  bacilli  can  be  found  microscopically,  may 
be  proved,  on  experimental  inoculation  into  animals,  to  be  still 
virulent.  In  such  cases  the  bacilli  may  be  present  in  numbers 
so  small  as  to  escape  observation,  or  it  may  be  that  their  spores 
only  are  present.  In  subacute  lesions,  with  well-formed  tubercle 
follicles  and  little  caseation,  the  bacilli  are  generally  scanty. 
They  are  most  numerous  in  acute  lesions,  especially  where 
caseation  is  rapidly  spreading,  for  example,  in  such  conditions 
as  caseous  catarrhal  pneumonia  (Fig.  90),  acute  tuberculosis  of 
the  spleen  in  children,  which  is  often  attended  with  a  good  deal 
of  rapid  caseous  change,  etc.  In  acute  miliary  tuberculosis  a 
few  bacilli  can  generally  be  found  in  the  centre  of  the  follicles ; 
but  here  they  are  often  much  more  scanty  than  one  would  ex- 
pect The  tubercle  bacillus  is  one  which  not  only  has  compara- 


DISTRIBUTION    OF   THE    BACILLI. 


247 


tively  slow  growth,  but  retains  its  form  and  staining  power  for  a 

much  longer  period  than  most  organisms.     This  is  true  of  the 

bacilli  both  in 

cultures      and 

also     in      the 

tissues. 

As  regards 
their  position 
in  the  tissues, 
the  bacilli  are 
usually  scat- 
tered irregu-  f^f^M  .•*.  FVJ 
larly  or  in 
small  groups 
amongst  the 
cells  or  gran- 
ular material. 
Most  of  the 
bacilli  lie  free, 
and  their  oc- 
currence with- 
in the  cells  is 
relatively  un- 
common, there 

being  in  this  respect  a  contrast  to  what  is  seen  in  the  lesions  in 
leprosy.  Occasionally  we  find  them  within  the  giant-cells, 
in  which  they  may  be  arranged  in  a  somewhat  radiate  manner 
at  the  periphery,  occasionally  also  in  epithelioid  cells  and  in 
leucocytes ;  but  these  are  by  no  means  frequent  sites  in  the 
human  subject. 

The  above  statements,  however,  apply  only  to  tuberculosis 
in  the  human  subject,  and  even  in  this  case  there  are  exceptions. 
In  the  ox,  on  the  other  hand,  the  presence  of  tubercle  bacilli 
within  giant-cells  is  a  very  common  occurrence ;  and  it  is  also 
common  to  find  them  in  considerable  numbers  scattered  irregu- 
larly throughout  the  cellular  connective  tissue  of  the  lesions, 
even  when  there  is  little  or  no  caseation  present  (Fig.  91). 

In  tuberculosis  in  the  horse,  and  in  avian  tuberculosis,  the 
numbers  of  bacilli  may  be  enormous,  even  in  lesions  which  are 
not  specially  acute;  and  considerable  variation  both  in  their 


FIG.  90.  —  Tubercle  bacilli  in  section  of  human  lung  in  acute 
phthisis.  The  bacilli  are  seen  lying  singly,  and  also  in  large  masses 
to  left  of  field.  The  pale  background  is  formed  by  caseous  material. 
Stained  with  carbol-fuchsin  and  Bismarck-brown.  X  1000. 


248 


TUBERCULOSIS. 


> 

\ 


tf 


a , 


number  and  in  their  site  is  met  with  in  tuberculosis  of  other 

animals. 

%  I  n       dis- 

charges   from 

tubercular    le- 

H  l  u-  u 

«'.^       %  sions       which 

/  are  breaking 

down,  tuber- 
cle bacilli  are 
usually  to 
be  found.  In 
the  sputum  of 
phthisical  pa- 
tients their 
presence  can 
be  demon- 
strated  almost 
invariably  at 
some  period, 
and  sometimes 
their  numbers 
are  very  large 
(for  method  of 

staining  see  p.  104).     Several  examinations  may,  however,  require 

to  be  made ;  this  should  always  be  done  before  any  conclusion 

as  to  the  non-tubercular  nature 

of  a  case  is  come  to.     In  cases 

of   genito-urinary   tuberculosis 

they  are  usually  present  in  the 

urine ;   but   as  they  are  much 

diluted    it   is    difficult    to    find 

them   unless  a  very   complete 

formation  of  deposit  is  allowed 

to  take  place.     This  deposit  is 

examined  in  the  same  way  as 

the  sputum.      It  is,   however, 

much  easier  to  obtain  their  sep- 


FIG.  91.  —  Tubercle  bacilli  in  giant-cells,  showing  the  radiate 
arrangement  at  the  periphery  of  the  cells.  Section  of  tubercular 
udder  of  cow.  Stained  with  carbol-fuchsin  and  Bismarck-brown. 
X  loco. 


aration  by  means  Of  the  Cen- 
trifuge.  If  this  method  is 
employed,  bacilli  Can  Usually 


FIG.  92.  —  Tubercle  bacilli  in  urine  ;  show- 
ing  one  of  the  characteristic  clumps,  in  which 

they  often  occur.    Stained  with  carbol-fuchsin 
and  methylene-blue.     Xiooo. 


EXPERIMENTAL   INOCULATION. 


249 


be  detected,  though  sometimes  their  number  may  be  very  small ; 
here,  especially,  repeated  examinations  may  be  necessary.  The 
bacilli  often  occur  in  little  clumps,  as  shown  in  Fig.  92.  In 
tubercular  ulceration  of  the  intestine  their  presence  in  the 
faeces  may  be  demonstrated,  as  was  first  shown  by  Koch ;  but 
in  this  case  their  discovery  is  usually  of  little  importance,  as 
the  intestinal  lesions,  as  a  rule,  occur  only  in  advanced  stages 
when  diagnosis  is  no  longer  a  matter  of  doubt. 

Experimental  Inoculation.  —  Tuberculosis  can  be  artificially 
produced  in  animals  by  infection  in  a  great  many  different  ways 
—  by  injection  of  the  bacilli  into  the  subcutaneous  tissue,  into  the 
peritoneum,  into  the  anterior  chamber  of  the  eye,  into  the  veins  ; 
by  feeding  the  animals  with  the  bacilli;  and,  lastly,  by  making 
them  inhale  the  bacilli  suspended  in  the  air. 

The  exact  result,  of  course,  varies  in  different  animals  and 
according  to  the  method  of  inoculation,  but  we  may  state  gen- 
erally that  when  introduced  into  the  tissues  of  a  susceptible 
animal,  the  bacilli  produce  locally  the  lesions  above  described, 
terminating  in  caseation ;  that  there  occurs  a  tubercular  affec- 
tion of  the  neighbouring  lymphatic  glands,  and  that  lastly 
there  may  be  a  rapid  extension  of  the  bacilli  to  other  organs 
by  the  blood  stream  and  the  production  of  general  tuberculosis. 
Of  the  animals  used  for  the  purpose,  the  guinea-pig  is  most 
susceptible. 

When  a  guinea-pig  is  inoculated  subcutaneously  with  tubercle 
bacilli  from  a  culture,  or  with  material  containing  them,  such 
as  phthisical  sputum,  a  local  swelling  gradually  forms  which 
is  usually  well  marked  about  the  tenth  day.  This  swelling 
becomes  softened  and  caseous,  and  may  break  down,  leading 
to  the  formation  of  an  irregularly  ulcerated  area  with  caseous 
lining.  The  lymphatic  glands  in  relation  to  the  parts  can  gen- 
erally be  found  to  be  enlarged  and  of  somewhat  firm  consist- 
ence, about  the  end  of  the  second  or  third  week.  Later,  in 
them  also  caseous  change  occurs,  and  a  similar  condition  may 
spread  to  other  groups  of  glands  in  turn,  passing  also  to  those 
on  the  other  side  of  the  body.  During  the  occurrence  of  these 
changes,  the  animal  loses  weight,  gradually  becomes  cachectic, 
and  ultimately  dies,  sometimes  within  six  weeks,  sometimes  not 
for  two  or  three  months.  Post  mortem,  in  addition  to  the  local 
and  glandular  changes,  an  acute  tuberculosis  is  usually  present, 


250  TUBERCULOSIS. 

the  spleen  being  specially  affected.  This  organ  is  swollen,  and 
is  studded  throughout  by  numerous  tubercle  nodules,  which 
may  be  minute  and  grey,  or  larger  and  of  a  yellowish  tint.  If 
death  has  been  long  delayed,  calcification  may  have  occurred 
in  some  of  the  nodules.  Tubercle  nodules,  though  rather 
less  numerous,  are  also  present  in  the  liver  and  in  the  lungs, 
the  nodules  in  the  latter  organs  being  usually  of  smaller  size 
though  occasionally  in  large  numbers.  The  extent  of  the  gen- 
eral infection  varies ;  sometimes  the  chronic  glandular  changes 
constitute  the  outstanding  feature. 

Intraperitoneal  injection  of  pure  cultures  produces  a  local 
lesion  in  the  form  of  an  extensive  tubercular  infiltration  and 
thickening  of  the  omentum,  sometimes  attended  with  acute 
tubercles  all  over  the  peritoneum.  There  is  a  caseous  enlarge- 
ment of  the  retroperitoneal  and  other  lymphatic  glands,  and 
later  there  may  be  a  general  tuberculosis.  Intravenous  injec- 
tion produces  a  typical  acute  tuberculosis,  the  nodules  being 
usually  more  numerous  and  of  smaller  size,  while  death  follows 
more  rapidly,  the  larger  the  numbers  of  bacilli  injected.  Guinea- 
pigs,  when  fed  with  tubercle  bacilli,  or  with  sputum  or  portions 
of  tissue  containing  them,  readily  contract  an  intestinal  form 
of  tuberculosis,  lesions  being  present  in  the  lymphoid  tissue  of 
the  intestines,  in  the  mesenteric  glands,  and  later  in  the  internal 
organs. 

Rabbits  are  less  susceptible  than  guinea-pigs,  and  in  them 
the  effects  of  subcutaneous  inoculation  are  very  variable ;  some- 
times the  lesions  remain  local,  sometimes  a  general  tuberculosis 
is  set  up.  Otherwise  the  reactions  are  much  of  the  same 
nature.  Dogs  are  much  more  highly  resistant,  but  tubercu- 
losis can  be  produced  in  them  by  intraperitoneal  injection  of 
pure  cultures  (Koch),  or  by  intravenous  injection  (Maffucci). 
In  the  latter  case  there  results  an  extensive  eruption  of  minute 
miliary  tubercles.  Tuberculosis  can  also  be  easily  produced 
in  susceptible  animals  by  making  them  inhale  the  bacilli. 

Varieties  of  Tuberculosis,  i.  Human  and  Bovine  Tubercu- 
losis. —  Although  variations  in  the  virulence  of  the  tubercle 
bacilli  from  different  sources  had  been  repeatedly  observed,  no 
systematic  comparison  had  up  till  recently  been  made,  and  it 
was  generally  accepted  that  all  mammalian  tuberculosis  was  due 
to  the  same  organisms,  and  in  particular  that  tuberculosis  could 


HUMAN   AND    BOVINE  TUBERCULOSIS.  251 

be  transmitted  from  the  ox  to  the  human  subject.  The  matter 
has  become  one  of  special  interest  owing  to  Koch's  address 
at  the  Tuberculosis  Congress  in  1901,  in  which  he  stated  his 
conclusion  that  human  and  bovine  tuberculosis  are  practically 
distinct,  and  that  if  a  susceptibility  of  the  human  subject  to  the 
latter  really  exists,  infection  is  of  very  rare  occurrence,  —  so 
rare  that  it  is  not  advisable  to  take  any  measures  against  it. 
Previously  to  this,  Theobald  Smith  had  pointed  out  differences 
between  mammalian  and  bovine  tubercle  bacilli,  the  most  im- 
portant being  that  the  latter  possess  a  much  higher  virulence 
to  the  guinea-pig,  rabbit,  and  other  animals,  and  in  particular  that 
human  tubercle  bacilli,  on  inoculation  into  oxen,  produce  either 
no  disease  or  only  local  lesions  without  any  dissemination.  He 
also  found  that  the  bovine  bacilli  on  cultivation  grow  less  vigor- 
ously for  a  time,  and  tend  to  be  shorter  and  straighter  than  the 
human  bacilli.  Koch's  conclusions  were  based  chiefly  on  the 
result  of  his  inoculations  in  the  bovine  species  with  human 
tubercle  bacilli,  the  result  being  confirmatory  of  Smith's,  and, 
secondly,  on  the  supposition  that  infection  of  the  human  subject 
through  the  intestine  is  of  very  rare  occurrence.  With  regard 
to  this  opinion,  we  must  disagree  with  Koch,  as  in  our  experi- 
ence there  is  considerable  evidence  that  in  young  subjects  the 
intestinal  canal  is  a  comparatively  common  path  of  entrance ; 
and,  moreover,  the  presence  of  pulmonary  lesions  does  not  prove 
that  infection  has  occurred  by  inhalation,  as  in  many  cases  the 
pulmonary  lesions  are  secondary  to  those  in  the  bronchial 
glands,  whilst  the  infection  of  the  cervical  or  mesenteric  glands 
is  of  still  older  standing.  There  may  also  be  infection  of  the 
mesenteric  glands  without  actual  lesions  in  the  intestine.  That 
the  ox  is  little  susceptible  to  human  bacilli  may  be  accepted,  but 
it  does  not  follow  that  the  converse  is  true,  namely,  that  the 
human  subject  cannot  be  infected  from  the  bovine  species, 
seeing  that  bovine  tubercle  bacilli  have  been  found  to  have 
a  greater  virulence  for  all  animals  tested  than  bacilli  from 
the  human  subject.  Moreover,  there  are  cases,  notably  those 
recorded  by  Ravenel,  in  which  direct  inoculation  of  the  human 
subject  with  bovine  tubercle  has  occurred.  Even  if  the  human 
subject  is  little  susceptible  to  bovine  tuberculosis,  it  is  quite 
likely,  in  view  of  the  large  proportion  of  young  subjects  ex- 
posed to  infection,  that  the  number  of  cases  of  tuberculosis  pro- 


252  TUBERCULOSIS. 

duced  in  this  way  is  by  no  means  small.  And  furthermore, 
although  the  ox  is  little  susceptible  to  human  tubercle  bacilli, 
tuberculosis  with  general  infection  has  been  produced  in  calves 
by  means  of  them  on  more  than  one  occasion.  Such  a  result 
has  been  obtained  by  Ravenel,  and  also,  in  this  country,  by 
Delepine.  There  are  also  facts  which  go  to  show  that  tubercle 
bacilli  cultivated  from  lesions  in  young  children  have  a  higher 
degree  of  virulence  for  animals  than  those  obtained  from  adults  ; 
that  is,  they  resemble  more  the  bovine  tubercle  bacilli ;  this 
is  what  one  might  expect  if  the  bacilli  in  question  had  come 
comparatively  recently  from  the  tissues  of  the  ox.  As  at  pres- 
ent the  subject  is  still  under  investigation  in  this  and  other 
countries,  it  would  not  be  justifiable  to  dogmatise,  but  in  the 
meantime,  we  see  no  sufficient  reason  to  depart  from  the  view 
entertained  up  to  this  time,  that  the  tubercle  bacilli  infecting 
mammals  are  of  one  and  the  same  species,  though  differences  in 
virulence  obtain,  and  that  milk  containing  tubercle  bacilli  is 
a  highly  important  source  of  infection  to  the  human  subject.  It 
may  also  be  added  that  tubercle  bacilli  obtained  from  other 
mammals  than  the  ox  generally  correspond  more  closely,  as 
regards  their  virulence  or  inoculation,  with  bovine  than  with 
human  bacilli. 

2.  Avian  Tuberculosis.  —  In  the  tubercular  lesions  in  birds 
there  are  found  bacilli  which  correspond  in  their  staining  reac- 
tions and  in  their  morphological  characters  with  those  in  mam- 
mals, but  differences  are  observed  in  cultures,  and  also  on 
experimental  inoculation.  These  differences  were  first  de- 
scribed by  Maffucci  and  by  Rivolta,  but  special  attention  was 
drawn  to  the  subject  by  a  paper  read  by  Koch  at  the  Internar 
tional  Medical  Congress  in  1890.  Koch  stated  that  he  had 
failed  to  change  the  one  variety  of  tubercle  bacillus  into  the 
other,  though  he  did  not  conclude  therefrom  that  they  were 
quite  distinct  species.  The  following  points  of  difference  may 
be  noted. 

On  glycerin  agar  and  on  serum,  the  growth  of  tubercle  bacilli  from  birds 
is  more  luxuriant,  has  a  moister  appearance  (Fig.  89,0),  and,  moreover,  takes 
place  at  a  higher  temperature,  43.5°  C.,  than  is  the  case  with  ordinary  tubercle 
bacilli.  Experimental  inoculation  brings  out  even  more  distinct  differences. 
Tubercle  bacilli  derived  from  the  human  subject,  for  example,  when  injected 
into  birds,  usually  fail  to  produce  tuberculosis,  whilst  those  of  avian  origin 
very  readily  do  so.  Birds  are  also  very  susceptible  to  the  disease  when  fed 


AVIAN    TUBERCULOSIS. 


253 


with  portions  of  the  organs  of  birds  containing  tubercle  bacilli,  but  they  can 
•consume  enormous  quantities  of  phthisical  sputum  without  becoming  tuber- 
cular (Straus,  Wurtz,  Nocard) .  No  doubt,  on  the  other  hand,  there  are  cases 
on  record  in  which  the  source  of  infection  of  a  poultry  yard  has  apparently 
been  the  sputum  of  phthisical  patients.  Again,  tubercle  bacilli  cultivated  from 
birds  have  not  the  same  effect  on  inoculation  of  mammals,  as  ordinary  tubercle 
bacilli.  When  guinea-pigs  are  inoculated  subcutaneously  they  usually  resist 
infection,  though  occasionally  a  fatal  result  follows.  In  the  latter  case,  usually 
no  tubercles  visible  to  the  naked  eye  are  found,  but  numerous  bacilli  may  be 
present  in  internal  organs,  especially  in  the  spleen,  which  is  much  swollen. 
Further,  intravenous  injection  even  of  large  quantities  of  avian  tubercle  bacilli, 
in  the  case  of  dogs,  leads  to  no  effect,  whereas  ordinary  tubercle  bacilli  pro- 
duce acute  tuberculosis.  [The  rabbit,  on  the  other  hand,  is  comparatively 
susceptible  to  avian  tuberculosis  (Nocard).] 

There  is,  therefore,  abundant  evidence  that  the  bacilli  derived 
from  the  two  classes  of  animals  show  important  differences,  and, 
reasoning  from  analogy,  we  might  infer  that  probably  the  human 
subject  also  would  be  little  susceptible  to  infection  from  avian 
tuberculosis.  The  question  remains,  are  these  differences  of  a 
permanent  character?  The  matter  seems  permanently  settled 
by  the  experiments  of  Nocard,  in  which  mammalian  tubercle 
bacilli  have  been  made  to  acquire  all  the  characters  of  those  of 
.avian  origin.  The  method  adopted  was  to  place  bacilli  from 
human  tuberculosis  in  small  collodion  sacs  containing  bouillon, 
and  then  to  insert  each  sac  in  the  peritoneal  cavity  of  a  fowl. 
The  sacs  were  left  in  situ  for  periods  of  from  four  to  eight  months. 
They  were  then  removed,  cultures  were  made  from  their  con- 
tents, fresh  sacs  were  inoculated  from  these  cultures  and  intro- 
duced into  other  fowls.  In  such  conditions  the  bacilli  are 
subjected  only  to  the  tissue  juices,  the  wall  of  the  sac  being 
impervious  both  to  bacilli  and  to  leucocytes,  etc.  After  one 
sojourn  of  this  kind,  and  still  more  so  after  two,  the  bacilli  are 
found  to  have  acquired  some  of  the  characters  of  avian  tubercle 
bacilli,  but  are  still  non-virulent  to  fowls.  After  the  third  so- 
journ, however,  they  have  acquired  this  property,  and  produce  in 
fowls  the  same  lesion  as  bacilli  derived  from  avian  tuberculosis. 
It  therefore  appears  that  the  bacilli  of  avian  tuberculosis  are 
not  a  distinct  and  permanent  species,  but  a  variety  which  has 
been  modified  by  growth  in  the  tissues  of  the  bird.  Evidently 
also  there  are  degrees  of  this  modification  according  to  the 
period  of  time  during  which  the  bacilli  have  passed  from  bird  to 


254  TUBERCULOSIS. 

bird,  as  in  some  cases  inoctilation  with  tubercle  ba'cilli  of  avian 
origin  has  produced  ordinary  tubercle  nodules  in  guinea-pigs 
(Courmont  and  Dor).  We  may  add  that  tuberculin  prepared 
from  avian  tubercle  bacilli  has  the  same  action  as  the  ordinary 
tuberculin. 

3.  Tuberculosis  in  the  Fish.  —  Bataillon,  Dubard,  and  Terre 
cultivated  from  a  tubercle-like  disease  in  a  carp,  a  bacillus  which, 
in  staining  reaction  and  microscopic  characters,  closely  agrees 
with  the-  tubercle  bacillus.  The  lesion  with  which  it  was  asso- 
ciated was  an  abundant  growth  of  granulation  tissue  in  which 
numerous  giant-cells  were  present.  It  forms,  however,  luxuriant 
growth  at  the  room  temperature,  the  growth  being  thick  and 
moist  like  that  of  avian  tubercle  bacilli  (Fig.  94,  c).  Growth 
does  not  occur  at  the  body  temperature,  though  by  gradual 
acclimatisation  a  small  amount  of  growth  has  been  obtained  up 
to  36°  C.  Furthermore,  the  organism  appears  to  undergo  no 
multiplication  when  injected  into  the  tissues  of  mammals,  and 
attempts  to  modify  this  characteristic  have  so  far  been  unsuc- 
cessful. An  interesting  fact,  however,  in  connection  with  this 
subject  is  that  it  has  been  found  possible  to  alter  the  conditions 
of  growth  of  the  tubercle  bacillus  from  the  human  subject  so 
that  it  flourishes  at  lower  temperature  than  normal.  This  has 
been  effected  by  allowing  it  to  remain  for  some  time  in  the 
tissues  of  the  frog,  its  optimum  temperatures  of  growth  after  a. 
time  being  28°-3O°  C.  and  Moeller,  by  a  similar  proceeding  in 
the  case  of  the  blindworm,  produced  marked  modification,  so 
that  the  organism  no  longer  grew  above  28°  C.,  whereas  it  flour- 
ished at  the  room  temperature.  Dubard  also  states  that  he  has 
succeeded  in  making  the  tubercle  bacillus  from  a  human  source 
assume  the  characters  of  the  bacillus  of  fish  tuberculosis. 

All  the  above  facts  taken  together  indicate  that  tubercle 
bacilli  may  become  modified  in  relative  virulence  and  in  condi- 
tions of  growth  by  sojourn  in  the  tissues  of  various  animals. 
This  modification  appears  slight,  though  of  definite  character  in 
the  case  of  bovine  tuberculosis,  more  distinct  in  the  case  of 
avian  tuberculosis,  and  much  more  marked,  if  not  permanent, 
in  the  case  of  fish  tuberculosis,  that  is,  of  course,  in  their  rela- 
tions to  the  bacilli  from  the  human  subject. 

Other  Acid-fast  Bacilli.  —  Within  recent  years  a  number  of 
bacilli  presenting  the  same  staining  reaction  as  the  tubercle 


OTHER   ACID-FAST   BACILLI. 


255 


bacilli  have  been  discovered.  Such  bacilli  have  a  compara- 
tively wide  distribution  in  nature,  as  they  have  been  obtained 
from  various  species  of  grass,  from  butter  and  milk,  from  manure, 
and  from  the  surfaces  of  animal  bodies.  Microscopically,  they 
agree  more  or  less  closely  with  tubercle  bacilli,  though  many 
are  shorter  and  plumper;  many  of  them  show  filamentous  and 
branching  forms  under  certain  conditions  of  culture.  Moreover, 
on  injection,  they  produce  granulation-tissue  modules  which  may 
closely  resemble  tubercles,  although  on  the  whole  there  is  a 
greater  tendency  to  softening  and  suppuration,  and  usually  they 
are  localised  at  the  site  of  inoculation.  The  most  important 
point  of  distinction  is  the  fact  that  their  multiplication  on  arti- 
ficial media  is  much  more  rapid,  growth  usually  being  visible 
within  forty-eight  hours  and  often  within  twenty-four  hours. 
Moreover,  in  most  instances,  growth  occurs  at  the  room  tem- 
perature. The  general  character  of  the  cultures  in  this  group  is 
a  somewhat  irregular  layer,  often  with  wrinkled  surface,  dry  or 
moist  in  appearance,  and  varying  in  tint  from  white  to  yellow  or 
reddish  brown.  The  number  of  such  organisms  is  constantly  being 
added  to,  but  the  following  may  be  mentioned  as  examples  :  — 

Moeller^s  Grass  Bacilli,  /.  and  II.  —  The  former  was  found  in  infusions 
of  Timothy-grass  (Phleum  pratense).     It  is  extremely  acid-fast,  morphologi- 
cally resembles  the  tubercle  bacillus, 
and  in  cultures  may  show  club  forma- 
tion   and    branching.      The    lesions  ^^          /*   * 
produced  closely  resemble  tubercles. 
The    colonies,    visible    in    thirty-six        ,4  £» 

i  i      iM  J       r  •    i_  4^   if 

hours,  are  scale-like  and  of  greyish-  /   <»  ?     ,  _  j 

white  colour.     Moeller's  bacillus  II.  |  '/(  A  # 

was  obtained  from  the  dust  of  a  hay-  -  ***Vj/*l>*  f^ 

loft.     The  colonies  at  first  are  moist  fej  &  .*>/ 

gjCS»"i        #        H  *     / 

and   somewhat    tenacious,  but  after-      V;|j  \f      / 

wards  run  together,  and  are  of  a  dull  *• 

yellowish    colour    (Fig.    93).      The  -Ai       "  ^\f^          1  \    *  **•* 

general  results  of  inoculation  resemble  *™""*^»j*.% 

those  of  grass  bacillus  I.  but  are  less  "••/ 

marked.      Moeller    also    obtained    a 

similar  organism  from  milk.     He  also  Fia  93—  Moeller's  Timothy-grass  bacil- 

lus.    From  a  culture  on  agar.     Stained  with 

discovered  a  third  acid-fast  bacillus     carbol-fuchsin,  and  treated  with  20  per  cent 
which    obtained    from    manure    and     sulphuric  acid,    x  1000. 
therefore  called   the   "  Mistbacillus " 

(dung  bacillus) .  This  organism  has  analogous  characters,  though  presenting 
minor  differences.  It  also  has  pathogenic  effects. 


256 


TUBERCULOSIS. 


FIG.  94.  —  Cultures  of  acid-fast  bacilli 
grown  at  room  temperature. 

(«)    Moeller's  Timothy-grass  bacillus  I. 
(6)    The   Petri-Rabinowitsch   butter-bacillus. 
(c)    Bacillus  of  fish  tuberculosis. 


Petri  and  Rabinowitsch  independently  cultivated  an  acid-fast  bacillus  from 

butter  ("butter  bacillus"),  in  which 
it  occurs  with  comparative  frequency. 
This  organism  resembles  the  tubercle 
bacillus,  although  it  is  on  the  whole 
shorter  and  thicker.  Its  lesions  closely 
resemble  tuberculosis,  especially  when 
injection  of  the  organism  is  made  into 
the  peritoneal  cavity  of  guinea-pigs, 
along  with  butter,  —  the  method  usually 
adopted  in  searching  for  tubercle  bacilli 
in  butter.  This  organism  produces  pretty 
rapidly  a  wrinkled  growth  (Fig.  94,  b)  not 
unlike  that  of  Moeller's  grass  bacillus  II. 
Korn  also  has  obtained  other  two  bacilli 
from  butter  which  he  holds  to  be  distinct 
from  one  another  and  from  Rabinowitsch 's 
bacillus.  The  points  of  distinction  are 
of  a  minor  character.  Other  more  or 
less  similar  bacilli  have  been  cultivated 
by  Tobler,  Coggi,  and  others.1 
Smegma  Bacillus.  —  This  organism  is  of  importance,  as  in  form  and  stain- 
ing reaction  it  somewhat  resembles  the  tubercle  bacillus  and  may  be  mistaken 
for  it.  It  occurs  often  in  large  numbers  in  the  smegma  prasputiale  and  in  the 
region  of  the  external  genitals,  espe- 
cially where  there  is  an  accumulation 
of  fatty  matter  from  the  secretions. 
Morphologically  it  is  a  slender,  slightly 
curved  organism,  like  the  tubercle 
bacillus  but  usually  distinctly  shorter  \  ^  \ 

(Fig.  95).     Like  the  tubercle  bacillus 

it  stains  with  some  difficulty  and  resists  i    -^4*  **%  ^  ^, 

decolorisation    with    strong    mineral      ^~  v       ^       «*  ^*lx 

acids.  Most  observers  ascribe  the 
latter  fact  to  the  fatty  matter  with 
which  it  is  surrounded,  and  find  that 
if  the  specimen  is  treated  with  alcohol 
the  organism  is  easily  decolorised. 
Czaplewski,  however,  who  claims  to 
have  cultivated  it  on  various  media, 
finds  that  in  culture  it  shows  resist- 
ance to  decolorisation  both  with  alco- 
hol and  with  acids,  and  considers,  therefore,  that  the  reaction  is  not  due  to 
the  surrounding  fatty  medium.  We  have  found  that  in  smegma  it  can  be 
readily  decolorised  by  a  minute's  exposure  to  alcohol  after  the  usual  treat- 

1  For  further  details  on  this  subject,  vide  Potet,  "  Etudes  sur  les  bacilles  dites 
acidophiles."     Paris,  1902. 


r- 


FIG.  95. — Smegma  bacilli.  Film  prep- 
aration of  smegma.  Ziehl-Neelsen  stain. 
X  looo. 


VALIDITY   OF   THE   STAINING   REACTION.  257 

ment  with  sulphuric  acid,  or  by  using  the  acid-alcohol  decoloriser,  and  thus 
can  be  readily  distinguished  from  the  tubercle  bacillus.  We,  moreover,  believe 
that  minor  points  of  difference  in  the  microscopic  appearances  of  the  two 
organisms  are  quite  sufficient  to  make  the  experienced  observer  suspicious  if 
he  should  meet  with  the  smegma  bacillus  in  urine,  and  lead  him  to  apply  the 
decolorising  test.  Difficulty  will  only  occur  when  a  few  scattered  bacilli 
retaining  the  fuchsin  occur. 

There  doubtless  occur  in  smegma  distinct  acid-resisting  bacterial  species. 
One  of  these,  cultivated  by  Laser,  Czaplewski,  and  Neufeld,  morphologically 
resembles  the  diphtheria  bacillus,  and  loses  its  acid-resisting  property  in  arti- 
ficial cultures.  The  bacillus  cultivated  from  smegma  by  A.  Moeller  by  the  aid 
of  human  serum  is  probably  identical  with  the  genuine  smegma  bacillus  first 
described  by  Tavel,  Alvarez,  and  Matterstock,  and  resists  decolorisation  by 
acids  even  after  prolonged  artificial  cultivation.  This  latter  bacillus  closely 
resembles  the  tubercle  bacillus  in  morphology,  but  it  grows  at  lower  tempera- 
tures in  ordinary  media  and  is  non-pathogenic. 

Cowie  has  recently  found  that  acid-fast  bacilli  are  of  common  occurrence 
in  the  secretions  of  the  external  genitals,  mammae,  etc.,  in  certain  of  the  lower 
animals,  and  that  these  organisms  vary  in  appearance.  He  considers  that  the 
term  "  smegma  bacillus  "  probably  represents  a  number  of  allied  species. 

Validity  of  the  staining  reaction. — The  question  may  be 
asked  —  Do  these  results  modify  the  validity  of  the  staining 
reaction  of  tubercle  bacilli  as  a  means  of  diagnosis  ?  The  source 
of  any  acid-fast  bacilli  in  question  is  manifestly  of  importance, 
and  it  may  be  stated  that  when  these  have  been  obtained  from 
some  source  outside  the  body,  or  where  contamination  from  with- 
out has  been  possible,  their  recognition  as  tubercle  bacilli  cannot 
be  established  by  microscopic  examination  alone.  In  the  case  of 
material  coming  from  the  interior  of  the  body,  however,  sputum, 
etc.,  the  condition  must  be  looked  on  as  different,  and  although  an 
acid-fast  bacillus  (not  tubercle)  has  been  found  by  Rabinowitsch 
in  a  case  of  pulmonary  gangrene  we  have  no  sufficient  data  for 
saying  that  acid-fast  bacilli  other  than  the  tubercle  bacillus  flourish 
within  the  tissues  of  the  human  body  except  in  such  rare  in- 
stances as  to  be  practically  negligible.  (To  this  statement  the 
case  of  the  leprosy  bacillus  is  of  course  an  exception.)  Accord- 
ingly, up  till  now,  the  microscopic  examination  of  sputum,  etc., 
cannot  be  said  to  have  its  validity  shaken,  and  we  have  the 
results  of  enormous  clinical  experience  that  such  examination 
is  practically  of  unvarying  value.  Nevertheless  the  facts  es- 
tablished with  regard  to  other  acid-fast  bacilli  must  be  kept 
carefully  in  view,  and  great  care  must  be  exercised  when  only 


258  TUBERCULOSIS. 

one  or  two  bacilli  are  found,  especially  if  they  deviate  in  their 
morphological  characters  from  the  tubercle  bacillus. 

Action  of  Dead  Tubercle  Bacilli.  —  The  remarkable  fact  has 
been  established  by  independent  investigators  that  tubercle  bacilli 
in  the  dead  condition,  when  introduced  into  the  tissues  in  suffi- 
cient numbers,  can  produce  tubercle-like  nodules.  Prudden  and 
Hodenpyl,  by  intravenous  injection  in  rabbits  of  cultures  ster- 
ilised by  heat,  produced  in  the  lungs  small  nodules  in  which 
giant-cells,  but  no  caseation,  were  occasionally  present,  and 
which  were  characterised  by  more  growth  of  fibrous  tissue  than 
in  ordinary  tubercle.  The  subject  has  been  very  fully  investi- 
gated with  confirmatory  results  by  Straus  and  Gamaleia,  who  find 
that,  if  the  number  of  bacilli  introduced  into  the  circulation  is 
large,  there  result  very  numerous  tubercle  nodules  with  well- 
formed  giant-cells,  and  occasionally  traces  of  caseation.  The 
bacilli  can  be  well  recognised  in  the  nodules  by  the  ordinary 
staining  method.  In  these  experiments  the  bacilli  were  killed 
by  exposure  to  a  temperature  of  115°  C.  for  ten  minutes  before 
being  injected.  Similar  nodules  can  be  produced  by  intraperi- 
toneal  injection.  Subcutaneous  injection,  on  the  other  hand, 
produces  a  local  abscess,  but  in  this  case  no  secondary  tubercles 
are  found  in  the  internal  organs.  Further,  in  many  of  the  ani- 
mals inoculated  by  the  various  methods  a  condition  of  marasmus 
sets  in  and  gradually  leads  to  a  fatal  result,  there  being  great 
emaciation  before  death.  These  experiments,  which  have  been 
confirmed  by  other  observers,  show  that  even  after  the  bacilli 
are  dead  they  preserve  their  staining  reactions  in  the  tissues  for 
a  long  time,  and  also  that  there  are  apparently  contained  in  the 
bodies  of  the  dead  bacilli  certain  substances  which  act  locally, 
producing  proliferative,  and  to  a  less  extent  degenerative, 
changes,  and  which  also  markedly  affect  the  general  nutrition. 
The  long  period  during  which  the  tubercle  bacillus,  as  compared 
with  other  organisms,  retains  even  when  dead  its  morphological 
and  staining  characters,  is  a  very  striking  feature.  S.  Stockman 
has  recently  found  that  an  animal  inoculated  with  large  numbers 
of  dead  tubercle  bacilli  afterwards  gives  the  tuberculin  reaction. 

Practical  Conclusions.  —  From  the  facts  above  stated  with 
regard  to  the  conditions  of  growth  of  the  tubercle  bacilli,  their 
powers  of  resistance,  and  the  paths  by  which  they  can  enter  the 
body  and  produce  disease  (as  shown  by  experiment),  the  man- 


SOURCES    OF   HUMAN   INFECTION.  259 

ner  by  which  tuberculosis  is  naturally  transmitted  can  be  readily 
understood.  Though  the  experiments  of  Sander  show  that 
tubercle  bacilli  can  multiply  on  vegetable  media  to  a  certain 
extent  at  warm  summer  temperature,  it  is  doubtful  whether  all 
the  conditions  necessary  for  growth  are  provided  to  any  extent 
in  nature.  At  any  rate,  the  great  multiplying  ground  of  tubercle 
bacilli  is  the  animal  body,  and  tubercular  tissues  and  secretions 
containing  the  bacilli  are  the  chief,  if  not  the  only,  means  by 
which  the  disease  is  spread.  The  tubercle  bacilli  leave  the 
body  in  large  numbers  in  the  sputum  of  phthisical  patients,  and 
when  the  sputum  becomes  dried  and  pulverised  they  are  set 
free  in  the  air.  Their  powers  of  resistance  in  this  condition 
have  already  been  stated.  As  examples  of  the  extent  to  which 
this  takes  place,  it  may  be  said  that  their  presence  in  the  air  of 
rooms  containing  phthisical  patients  has  been  repeatedly  dem- 
onstrated. Williams  placed  glass  plates  covered  with  glycerin 
in  the  ventilating  shaft  of  the  Brompton  Hospital,  and  after 
five  days  found,  by  microscopic  examination,  tubercle  bacilli  on 
the  surface,  whilst  Klein  found  that  guinea-pigs  kept  in  the 
ventilating  shaft  became  tubercular.  Cornet  produced  tuber- 
culosis in  rabbits  by  inoculating  them  with  dust  collected  from 
the  walls  of  a  consumptive  ward.  Tubercle  bacilli  are  also  dis- 
charged in  considerable  quantities  in  the  urine  in  tubercular 
disease  of  the  urinary  tract,  and  also  by  the  bowel  when  there 
is  tubercular  ulceration;  but,  so  far  as  the  human  subject  is 
concerned,  the  great  means  of  disseminating  the  bacilli  in  the 
outer  world  is  dried  phthisical  sputum,  and  the  source  of  danger 
from  this  means  can  scarcely  be  overestimated.  Every  phthisi- 
cal patient  ought  to  be  looked  upon  as  a  fruitful  source  of 
infection  to  those  around,  and  should  only  expectorate  on  to 
pieces  of  rag  which  are  afterwards  to  be  burnt,  or  into  special 
receptacles  which  are  to  be  then  sterilised  either  by  boiling  or 
by  the  addition  of  a  5  per  cent  solution  of  carbolic  acid. 

Another  great  source  of  infection  is  in  all  probability  the  milk 
of  cows  affected  with  tuberculosis  of  the  udder.  In  such  cases 
the  presence  of  tubercle  bacilli  in  the  milk  can  usually  be 
readily  detected  by  centrifugalising  it,  and  then  examining  the 
deposit  microscopically,  or  by  inoculating  an  animal  with  it.  As 
pointed  out  by  Woodhead  and  others,  the  milk  from  cows  thus 
affected  is  probably  the  great  source  of  tabes  mesenterica,  which 


260  TUBERCULOSIS. 

is  so  common  in  young  subjects.  In  these  cases  there  may 
be  tubercular  ulceration  of  the  intestine,  or  it  may  be  absent. 
Woodhead  found  that  out  of  127  cases  of  tuberculosis  in  children, 
the  mesenteric  glands  showed  tubercular  affection  in  100,  and 
that  there  was  ulceration  of  the  intestine  in  43.  It  is  especially 
in  children  that  this  mode  of  infection  occurs,  as  in  the  adult 
ulceration  of  the  intestine  is  rare  as  a  primary  affection,  though 
it  is  common  in  phthisical  patients  as  the  result  of  infection  by 
the  bacilli  in  the  sputum  which  has  been  swallowed.  There  is 
less  risk  of  infection  by  means  of  the  flesh  of  tubercular  animals, 
for,  as  stated  by  the  recent  Tuberculosis  Commission,  in  the  first 
place,  tuberculosis  of  the  muscles  of  oxen  being  very  rare,  there 
is  little  chance  of  the  bacilli  being  present  in  the  flesh  unless  the 
surface  has  been  smeared  with  the  juice  of  the  tubercular  organs, 
as  in  the  process  of  cutting  up  the  parts ;  and  in  the  second 
place,  even  when  present  they  will  be  destroyed  if  the  meat  is 
thoroughly  cooked. 

We  may  state,  therefore,  that  the  two  great  modes  of  infec- 
tion are  by  inhalation,  and  by  ingestion,  of  tubercle  bacilli.  By 
the  former  method  the  tubercle  bacilli  will  in  most  cases  be 
derived  from  the  human  subject ;  in  the  latter,  probably  from 
tubercular  cows,  though  inhaled  tubercle  bacilli  may  also  be 
swallowed,  and  contamination  of  food  by  tubercular  material 
from  the  human  subject  may  occur.  Both  in  inhalation  and 
in  ingestion,  tubercle  bacilli  may  lodge  about  the  pharynx  and 
thus  come  to  infect  the  pharyngeal  lymphoid  tissue,  tonsils,  etc., 
tubercular  lesions  of  these  parts  being  much  more  frequent  than 
was. formerly  supposed.  Thence  the  cervical  lymphatic  glands 
may  become  infected,  and  afterwards  other  groups  of  glands, 
bones,  or  joints,  and  internal  organs. 

Koch's  Tuberculin.  —  In  1890-91  Koch  introduced  a  sub- 
stance called  "tuberculin,"  as  a  curative  agent  for  tubercular 
affections.  He  stated  that  if  in  a  guinea-pig  suffering  from 
the  effects  of  a  subcutaneous  inoculation  with  tubercle  bacilli,  a 
second  subcutaneous  inoculation  of  tubercle  bacilli  was  practised 
in  another  part  of  the  body,  superficial  ulceration  occurred 
in  the  primary  tubercular  nodule,  the  wound  healed,  and  the 
animal  did  not  succumb  to  tuberculosis.  This  reaction  was 
further  studied  by  means  of  the  above-mentioned  tuberculin, 
which  consisted  of  a  concentrated  glycerin  bouillon  culture  of 


KOCH'S   OLD   TUBERCULIN.  26l 

tubercle  in  which  the  bacilli  had  been  killed  by  heat.  It  con- 
tains the  dead  and  often  macerated  bacilli,  the  substances  in- 
destructible by  boiling  which  existed  in  these  bacilli,  non-volatile 
products  formed  by  them  from  the  food  material  when  alive, 
and  the  concentrated  remains  of  the  bouillon  and  glycerin.  The 
injection  of  .25  c.c.  of  tuberculin  into  a  healthy  man  causes,  in 
from  three  to  four  hours,  malaise,  tendency  to  cough,  laboured 
breathing,  and  moderate  pyrexia;  all  of  which  pass  off  in  twenty- 
four  hours.  The  injection  (the  site  of  the  injection  being  quite 
unimportant),  however,  of  .01  c.c.  into  a  tubercular  person  gives 
rise  to  similar  symptoms,  but  in  a  much  more  aggravated  form, 
and  in  addition  there  occurs  around  any  tubercular  focus  great 
inflammatory  reaction,  resulting  in  necrosis  and  a  casting  off 
of  the  tubercular  mass,  when  this  is  possible,  as  for  instance 
in  the  case  of  lupus.  The  bacilli  are,  it  was  shown,  not  killed 
in  the  process. 

Koch's  theory  of  the  action  of  the  substance  was  that  the  tubercle  bacillus 
ordinarily  secretes  a  body  having  a  necrotic  action  on  the  tissues.  When  this 
i§  injected  into  a  tubercular  patient,  the  proportion  present  round  a  tubercular 
focus  is  suddenly  increased,  inflammatory  reaction  takes  place  around,  and 
necrosis  of  the  spreading  margin  occurs  very  rapidly,  the  material  containing 
the  living  or  dead  bacilli  being  thrown  off  en  masse  instead  of  being  disin- 
tegrated piecemeal.  It  appears,  however,  that  this  explanation  may  not  be 
the  true  one,  for,  on  the  one  hand,  other  substances  besides  products  of  the 
tubercle  bacillus  may  give  rise  to  similar  effects  in  tubercular  animals,  and,  on 
the  other,  a  similar  reaction  can  take  place  in  other  diseases  where  there  is 
locally  in  the  body  a  deposit  of  new  tissue.  Matthes  has,  for  instance,  found 
that  albumoses  and  peptones  isolated  from  the  ordinary  peptic  digestion  of 
various  albumins  give  the  same  reaction  in  tubercular  guinea-pigs.  The 
injection  of  milk,  lactic  acid,  ricin,  all  give  a  similar  result.  Before  the  dis- 
covery of  tuberculin,  Gamaleia  had  found  that  tubercular  animals  were  very 
susceptible  to  the  toxins  of  the  vibrio  Metchnikovi,  and  later  Metchnikoff 
found  that  a  similar  susceptibility  existed  towards  the  toxins  of  the  bacillus  of 
fowl  cholera. 

The  hopes  which  the  introduction  of  tuberculin  raised  that 
a  curative  agent  against  tuberculosis  had  been  discovered  were 
soon  found  not  to  be  justified.  It  was  very  difficult  to  see  how 
the  necrosed  material  which  it  produced  and  which  contained 
the  still  living  bacilli  could  be  got  rid  of  either  naturally,  as 
would  be  necessary  in  the  case  of  a  small  tubercular  deposit  in 
a  lung  or  a  lymphatic  gland,  or  artificially,  as  in  a  complicated 
joint-cavity  where  surgical  interference  could  be  undertaken. 


262  TUBERCULOSIS. 

Not  only  so,  but  the  ulceration  which  might  be  the  sequel  of 
the  necrosis  appeared  to  open  a  path  for  fresh  infection.  Soon 
facts  were  reported  which  justified  these  criticisms.  Cases 
where  rapid  acute  tubercular  conditions  ensued  on  the  use  of 
tuberculin  were  reported,  and  in  a  few  months  the  treatment 
was  practically  abandoned.  The  conditions  in  guinea-pigs  on 
which  the  discovery  was  based  have  since  been  found  not  to 
be  of  universal  occurrence. 

The  Toxins  of  the  Tubercle  Bacillus.  —  The  fact  that  tuber- 
culin was  a  powerfully  toxic  agent  stimulated  the  study  of  its 
constituents  in  the  belief  that  among  these  the  toxins  of  the 
tubercle  bacillus  were  present.  It  was  found  to  contain  several 
albumoses,  alkaloids,  extractives,  and  inorganic  salts.  The 
albumoses  originated  a  tuberculin  reaction  in  tubercular  guinea- 
pigs,  but  as  has  been  stated  this  is  also  true  of  ordinary  albu- 
moses. It  may  be  stated  that  the  tuberculin  reaction  is  obtained 
with  the  products  of  the  growth  of  the  bacillus  in  a  non-proteid 
medium.  But  further,  a  similar  reaction  has  taken  place  when 
tuberculin  has  been  injected  into  persons  suffering  from  diseases 
other  than  tubercle,  e.g.  cancer,  sarcoma,  syphilis.  Further 
investigations  on  this  subject  are  thus  required.  The  toxins  of 
tubercle  are  thus  possibly  not  of  the  nature  of  albumoses.  Of 
their  real  nature  we  are  still  ignorant.  From  what  is  known,  it 
is  possible  that  they  do  not  to  any  great  extent  diffuse  out  into 
the  culture  media.  It  has  been  found  that  if  tubercle  cultures 
are  filtered  germ-free,  the  filtrated  does  not  give  such  a  marked 
tuberculin  reaction  as  the  unfiltered  fluid.  Maragliano  has 
found  that  such  a  fluid,  however,  causes  in  animals  lowering 
of  temperature  and  sweating,  and  further  that  if  it  is  heated  at 
1 00°  C.  it  now  gives  a  much  more  marked  tuberculin  reaction. 
It  is  thus  possible  that  more  than  one  toxic  body  may  be  formed 
by  the  tubercle  bacillus,  but  from  what  has  been  said  it  will  be 
realised  that  to  consider  the  occurrence  of  the  tuberculin  reac- 
tion as  indicating  the  presence  of  the  products  of  the  tubercle 
bacillus  is  not  at  present  justifiable. 

The  Use  of  Tuberculin  in  the  Diagnosis  of  Tuberculosis  in 
Cattle.  —  This  is  now  the  chief  use  to  which  tuberculin  is  put. 
In  cattle,  tuberculosis  may  be  present  without  giving  rise  to 
apparent  symptoms.  It  is  thus  important  from  the  point  of 
view  of  human  infection  that  an  early  diagnosis  should  be  made. 


KOCH'S    NEW   TUBERCULIN.  263 

The  method  is  applied  as  follows.  The  animals  are  kept  twenty- 
four  hours  in  their  byres  and  the  temperature  is  taken  every 
three  hours,  from  four  hours  before  the  injection  till  twenty-four 
after.  The  average  temperature  in  cattle  is  102.2°  F.  ;  30  to 
40  centigrammes  of  tuberculin  are  injected,  and  if  the  animal 
be  tubercular  the  temperature  rises  2°  or  3°  F.  in  eight  to  twelve 
hours  and  continues  elevated  for  ten  to  twelve  hours.  Bang, 
who  has  worked  most  at  the  subject,  lays  down  the  principle 
that  the  more  nearly  the  temperature  approaches  104°  F.  the 
more  reason  for  suspicion  is  there.  He  gives  a  record  of  280 
cases  where  the  value  of  the  method  was  tested  by  subse- 
quent post-mortem  examination.  He  found  that  with  proper 
precautions  the  error  was  only  3.3  per  cent.  The  method  is 
largely  practised  on  the  Continent,  and  ought  to  be  more  widely 
applied. 

Koch's  New  Tuberculin.  —  Koch  in  1897  published  the 
results  of  further  researches  on  tuberculosis.  These  consisted 
(i)  of  an  attempt  to  immunise  animals  against  the  tubercle 
bacillus  by  employing  its  intracellular  toxins ;  (2)  of  trying  to 
utilise  such  an  immunisation  to  aid  the  tissues  of  an  animal 
already  attacked  with  tubercle  the  better  to  combat  the  effects 
of  the  bacilli.  The  method  of  obtaining  the  intracellular 
toxins  was  as  follows.  Bacilli  from  young  virulent  cultures 
were  dried  in  vacuo,  and  disintegrated  with  an  agate  pestle  and 
mortar,  treated  with  distilled  water  and  centrifugalised.  The 
clear  fluid  was  decanted,  and  is  called  by  Koch  "  tuberculin  O." 
It  has  the  properties  of  the  ordinary  tuberculin.  The  remain- 
ing deposit  was  again  dried,  bruised,  treated  with  water  and 
centrifugalised,  the  clear  fluid  being  again  decanted,  and  this 
process  was  repeated  with  successive  residues  till  no  residue 
remained.  These  fluids  put  together  constitute  what  Koch 
calls  "tuberculin  R."  It  is  said  to  contain  the  substances 
present  in  the  bacilli,  which  are  insoluble  in  glycerin  and  only 
produce  a  tuberculin  reaction  in  large  doses.  When  injected 
into  animals  in  repeated  and  increasing  doses,  -5^-  mgrm.  being 
the  initial  dose,  it  is  said  to  produce  immunity  against  the 
'original  extract,  against  "tuberculin  O,"  and  against  living  and 
virulent  tubercle  bacilli;  but  regarding  this,  opinions  differ. 
Cases  of  early  phthisis  in  man  and  of  lupus  have  been  treated 
with  "tuberculin  R,"  no  dose  being  given  which  raises  the 


264  TUBERCULOSIS. 

temperature  more  than  .5°  F.  Though  cases  of  lupus  have 
been  recorded  in  which  improvement  has  taken  place,  little 
success  has  attended  the  use  of  this  substance  as  a  remedial 
agent. 

Immunisation  against  the  Tubercle  Bacillus  :  Anti-tubercular 
Serum.  —  Tuberculosis  differs  from  other  diseases,  against  which 
animals  can  be  immunised,  in  that  there  is  no  evidence  that  one 
attack  protects  against  a  second.  Further,  we  have  no  means 
of  obtaining  truly  attenuated  tubercle  bacilli.  Many  attempts 
at  immunisation  have,  however,  been  made.  It  has  been 
thought  by  some  that  the  tubercle  bacilli  from  so-called  scrofu- 
lous glands  are  less  virulent  than  those  say  from  phthisis,  but 
apparently  here  sufficient  attention  has  not  been  paid  to  the 
difference  of  the  numbers  of  bacilli  injected  in  each  case,  and 
this  appears  to  be  a  very  important  point.  Experiments  have 
also  been  brought  forward  which  appear  to  show  that  the  injec- 
tion of  bacilli  from  avian  tuberculosis  could  protect  the  dog 
against  bacilli  derived  from  man.  But  these  are  not  yet  conclu- 
sive. Further,  many  attempts  have  been  made  at  immunisation 
against  the  tubercle  bacillus  by  the  employment  of  its  toxic 
products.  The  most  successful  have  been  those  of  Maragliano. 
We  have  seen  that  this  author  distinguishes  between  the  toxic 
bodies  contained  in  the  bodies  of  the  bacilli  (which  withstand, 
unchanged,  a  temperature  of  100°  C.)  and  those  secreted  into 
the  culture  fluid  (which  are  destroyed  by  heat).  The  substance 
used  by  him  for  immunising  his  animals  consists  of  three  parts 
of  the  former  and  one  of  the  latter.  The  animals  employed  are 
the  dog,  the  ass,  the  horse.  The  serum  obtained  from  these  is 
capable  of  protecting  healthy  animals  against  an  otherwise  fatal 
dose  of  tuberculin,  but  very  little  importance  can  be  attached  to 
this  result.  Maragliano  does  not  appear  to  have  studied  the 
effects  of  this  serum  on  tubercular  animals,  but  it  has  been  tried 
in  a  great  number  of  cases  of  human  tuberculosis,  2  c.c.  being 
injected  subcutaneously  every  two  days.  Improvement  is  said 
to  have  taken  place  in  a  certain  proportion,  especially  of  mild 
non-febrile  cases. 

Methods  of  Examination.  —  ( i )  Microscopic  Examination.  — 
Tuberculosis  is  one  of  the  comparatively  few  diseases  in  which  a 
diagnosis  can  usually  be  definitely  made  by  microscopic  exami- 
nation alone.  In  the  case  of  sputum,  one  of  the  yellowish  frag- 


CULTIVATION   OF   TUBERCLE   BACILLUS.  265 

ments  which  are  often  present  ought  to  be  selected ;  dried  films 
are  then  prepared  in  the  usual  way  and  stained  by  the  Ziehl- 
Neelsen  method  (p.  104).  In  the  case  of  urine  or  other  fluids  a 
deposit  should  first  be  obtained  by  centrifugalising  a  quantity 
in  a  test-tube,  or  by  allowing  the  fluids  to  stand  in  a  tall  glass 
vessel  (an  ordinary  burette  is  very  convenient).  Film  prepara- 
tions are  then  made  with  the  deposit  and  treated  as  before.  If 
a  negative  result  is  obtained  in  a  suspected  case,  repeated  ex- 
amination should  be  undertaken.  To  avoid  risk  of  contamina- 
tion with  the  smegma  bacillus  the  meatus  of  the  urethra  should 
be  cleansed  and  the  urine  first  passed  should  be  rejected,  or  the 
urine  may  be  drawn  off  with  a  sterile  catheter.  As  stated  above 
it  is  only  exceptionally  that  difficulty  will  arise  to  the  experienced 
observer  from  this  cause.  (For  points  to  be  attended  to,  vide 

P-  257-) 

(2)  Inoculation. — The  guinea-pig  is  the  most  suitable  ani- 
mal.    If  the  material  to  be  tested  is  a  fluid  it  is  injected  subcu- 
taneously  or  into  the  peritoneum ;  if  solid  or  semi-solid  it  is 
placed  in   a  small  pocket  in  the  skin  or  it  may  be  thoroughly 
broken   up    in   sterile  water  or  other  fluid   and  the  emulsion 
injected.     By  this  method,  material  in  which  no  tubercle  bacilli 
can  be  found  microscopically  may  sometimes  be  shown  to  be 
tubercular. 

(3)  Cultivation.  —  Owing  to  the  difficulties  this  is   usually 
quite  impracticable  as  a  means  of  diagnosis,  and  it  is  also  un- 
necessary.    The  best  method  to  obtain  pure  cultures  is  to  pro- 
duce tuberculosis  in  a  guinea-pig  by  inoculation  with  tubercular 
material,  and  to  kill  the  animal  after  a  period  of  four  or  five 
weeks.     Then  with  portions  of  a  tubercular  organ,  e.g.  the  spleen, 
tubes  of  solidified  blood  serum  (preferably  that  of  the  dog,  ob- 
tained aseptically  and  coagulated  by  an  exposure  of  three  hours 
to  a  temperature  of  75°  C.  in  the  slanting  position)  are  to  be 
inoculated  under   the  strictest  aseptic  precautions.      The  por- 
tions of  tissue  should  be  moderately  small,  and  should  be  gently 
rubbed  over  the  surface  of  the  medium  and  brought  to  rest. 
The  tubes  are  then  to   be  incubated  at  37°  C.  for  a  week  or 
ten  days,  when  the  pieces  of  tissue  are  again  to  be  rubbed  over 
the  surface  of  the  medium,  turned  over,  and  once  more  incu- 
bated for  a  similar  period  of  time,  when  growth  may  possibly 
be  seen  in  isolated  spots  on  the  surface  of  the  serum  or  ex- 


266  TUBERCULOSIS. 

tending  beyond  the  edges  of  the  introduced  tissue.  It  is  to  be 
recommended  that  the  tubes  of  serum  be  kept  in  the  horizontal 
position  during  incubation,  lest  on  account  of  the  low  density  of 
the  medium,  the  upright  position  should  damage  the  extent  of 
surface  necessary  for  growth. 

(4)  Agglutination.  — Within  the  last  three  years  Arloing  and 
Courmont  announced  that  they  had  been  able  to  obtain  aggluti- 
nation of  the  tubercle  bacillus  with  the  blood  serum  of  tuber- 
culous patients,  in  a  manner  analogous  to  that  introduced  by 
Gruber  and  Widal  in  the  diagnosis  of  typhoid  fever.  The  tech- 
nique is  full  of  difficulties  and  the  reaction  is  not  often  obtained 
in  dilutions  of  I  in  25,  but  usually  much  lower,  as  I  in  10,  I  in  15. 
C.  Fraenkel,  Neisser,  Dieudonne,  Beck,  and  Rabinowitsch  have 
shown  that  the  reaction  is  unreliable,  being  often  absent  in 
patients  undoubtedly  tuberculous.  And  quite  recently  Koch 
has  demonstrated  that  the  reaction  may  appear  in  non-tuber- 
culous cases  in  dilutions  of  i  in  25,  or  in  one  case  (muscular 
rheumatism)  as  high  as  I  in  50,  and  he  fully  agrees  with  the 
others  above  cited  that  the  reaction  is  too  uncertain  to  be  of 
any  practical  value.  Furthermore,  it  is  of  interest  that  serum 
which  agglutinates  tubercle  bacilli  will  agglutinate  also  many  of 
the  other  acid-resisting  bacilli  which  for  this  and  other  reasons 
may  be  regarded  as  allied  to  B.  tuberculosis. 


CHAPTER  XL 
LEPROSY. 

LEPROSY  is  a  disease  of  great  interest,  alike  in  its  clinical  and 
pathological  aspects ;  whilst  from  the  bacteriological  point  of 
view  also,  it  presents  some  striking  peculiarities.  The  invariable 
association  of  large  numbers  of  characteristic  bacilli  with  all 
leprous  lesions  is  a  well-established  fact,  and  yet,  so  far,  at- 
tempts to  cultivate  the  bacilli  outside  the  body,  or  to  produce 
the  disease  experimentally  in  animals,  have  been  attended  with 
failure.  Leprosy,  so  far  as  is  known,  is  a  disease  which  is  con- 
fined to  the  human  subject,  but  it  has  a  very  wide  geographical 
distribution.  It  occurs  in  certain  parts  of  Europe — Norway, 
Russia,  Greece,  etc.,  but  is  commonest  in  Asia,  occurring  in 
Syria,  Persia,  etc.  It  is  prevalent  in  Africa,  being  especially 
found  along  the  coast,  in  the  Pacific  Islands,  in  the  warmer 
parts  of  North  and  South  America,  and  also  to  a  small  extent 
in  the  northern  part  of  North  America.  In  all  these  various 
regions  the  disease  presents  the  same  general  features,  and 
the  study  of  its  pathological  and  bacteriological  characters, 
wherever  such  has  been  carried  on,  has  yielded  similar  results. 

Pathological  Changes.  — Leprosy  is  characteristically  a  chronic 
disease,  in  which  there  is  a  great  amount  of  tissue  change,  with 
comparatively  little  necessary  impairment  of  the  general  health. 
In  other  words,  the  local  effects  of  the  bacilli  are  well  marked, 
often  extreme,  whilst  the  toxic  phenomena  are  proportionately  at 
a  minimum. 

There  are  two  chief  forms  of  leprosy.  The  one,  usually 
called  the  tubercular  form, — lepra  ttiberosa  or  tuberculosa,  —  is 
characterised  by  the  growth  of  granulation  tissue  in  a  nodular 
form  or  as  a  diffuse  infiltration  in  the  skin,  in  mucous  mem- 
branes, etc.,  great  disfigurement  often  resulting.  In  the  other 
form,  the  anaesthetic,  —  maculo-anaesthetic  of  Hansen  and  Looft, 
—  the  outstanding  changes  are  in  the  nerves,  with  consequent 
anaesthesia,  paralysis  of  muscles,  and  trophic  disturbances. 

267 


268 


LEPROSY. 


In  the  tubercular  form  the  disease  usually  starts  with  the 
appearance  of  erythematous  patches  attended  by  a  small  amount 
of  fever,  and  these  are  followed  by  the  development  of  small 
nodular  thickenings  in  the  skin,  especially  of  the  face,  of  the 
backs  of  hands  and  feet,  and  of  the  extensor  aspects  of  arms  and 
legs.  These  nodules  enlarge  and  produce  great  distortion  of 

the  surface,  so 
that,  in  the  case 
of  the  face,  an  ap- 
pearance is  pro- 
duced which  has 
been  described  as 
"leonine."  The 
thickenings  oc- 
cur chiefly  in  the 
cutis  (Fig.  96),  to 
a  less  extent  in 
the  subcutaneous 
tissue.  The  epi- 
thelium often  be- 
comes stretched 
over  them,  and 
an  oozing  sur- 
face becomes  de- 

FlG.  96.  —  Section  through  leprous  skin,  showing  the  masses  . 

of  cellular  granulation  tissue  in  the  cutis;  the  dark  points  are    VeiOped,Or actual 


clumps  of  bacilli  deeply  stained. 

Paraffin  section  ;  Ziehl-Neelsen  stain.     X  80. 


ulceration  may 
occur.  The  cor- 
nea and  other  parts  of  the  eye,  the  mucous  membrane  of 
the  mouth,  larynx,  and  pharynx,  may  be  the  seat  of  similar 
nodular  growths.  Internal  organs,  especially  the  spleen,  liver, 
and  testicles,  may  become  secondarily  affected.  In  all  situa- 
tions the  change  is  of  the  same  nature,  —  a  sort  of  chronic  in- 
flammatory condition  attended  by  abundant  formation  of  gran- 
ulation tissue,  nodular  or  diffuse  in  its  arrangement.  In  this 
tissue  a  large  proportion  of  the  cells  are  of  rounded  or  oval 
shape,  like  hyaline  leucocytes ;  a  number  of  these  may  be  of 
comparatively  large  size,  and  may  show  vacuolation  of  their  pro- 
toplasm and  a  vesicular  type  of  nucleus.  These  are  often  known 
as  "  lepra  cells."  Amongst  the  cellular  elements  there  is  a  vary- 
ing amount  of  stroma,  which  in  the  earlier  lesions  is  scanty  and 


THE   BACILLUS   OF   LEPROSY.  269 

delicate,  but  in  the  older  lesions  may  be  very  dense.  Periarteritis 
is  a  common  change,  and  very  frequently  the  superficial  nerves 
become  involved  in  the  nodules  and  undergo  atrophy.  The 
tissue  in  the  leprous  lesions  is  comparatively  vascular,  at  least 
when  young,  and,  unlike  tubercular  lesions,  never  shows  casea- 
tion.  Some  of  the  lepra  cells  may  contain  several  nuclei,  but 
we  do  not  meet  with  cells  resembling  in  their  appearance  tuber- 
cle giant-cells,  nor  does  an  arrangement  like  that  in  tubercle 
follicles  occur. 

In  the  ancesthetic  form  the  lesion  of  the  nerves  is  the  out- 
standing feature.  These  are  the  seat  of  diffuse  infiltrations  which 
lead  to  the  destruction  of  the  nerve  fibres.  In  the  earlier  stages, 
in  which  the  chief  symptoms  are  pains  along  the  nerves,  there 
occur  patches  on  the  skin,  often  of  considerable  size,  the  margins 
of  which  show  a  somewhat  livid  congestion.  Later,  these  patches 
become  pale  in  the  central  parts,  and  the  periphery  becomes 
pigmented.  There  then  follow  remarkable  series  of  trophic  dis- 
turbances in  which  the  skin,  muscles,  and  bones  are  especially 
involved.  The  skin  often  becomes  atrophied,  parchment-like, 
and  anaesthetic  ;  frequently  pemphigoid  bullae  or  other  skin 
eruptions  occur.  The  bones  become  atrophied,  and,  owing  to 
the  irregular  affection  of  the  muscles,  great  distortion  of  the 
extremities  may  result.  Partly  owing  to  injury  to  which  the  feet 
and  arms  are  liable  from  their  anaesthetic  condition,  and  partly 
owing  to  trophic  disturbances,  necrosis  and  separation  of  parts  are 
liable  to  occur.  In  this  way  great  distortion  results.  The  lesions 
in  the  nerves  are  of  the  same  nature  as  those  described  above, 
that  is,  they  are  the  result  of  a  chronic  inflammatory  process, 
but  the  granulation  tissue  is  less  in  amount,  and  has  a  greater 
tendency  to  undergo  cicatricial  contraction.  This  is  to  be 
associated  with  the  fact  that  the  bacilli  are  present  in  fewer 
numbers. 

Bacillus  of  Leprosy.  —  This  bacillus  was  first  observed  in 
leprous  tissues  by  Hansen  in  1871,  and  was  the  subject  of  several 
communications  by  him  in  1874  and  later.  Further  researches, 
first  by  Neisser  in  1879,  and  afterwards  by  observers  in  various 
parts  of  the  world,  agreed  in  their  main  results,  and  confirmed 
the  accuracy  of  Hansen's  observations.  The  bacilli  as  seen  in 
scrapings  of  ulcerated  leprous  nodules,  or  in  sections,  have  the 
following  characters.  They  are  thin  rods  of  practically  the  same 


2/0 


LEPROSY. 


size  as  tubercle  bacilli,  which  they  also  resemble  both  in  appear- 
ance and  in  staining  reaction.  They  are  straight  or  slightly 
curved,  and  usually  occur  singly,  or  two  may  be  attached  end 
to  end ;  but  they  do  not  form  chains.  When  stained  they  may 
have  a  uniform  appearance,  or  the  protoplasm  may  be  frag- 
mented, so  that 
they  appear  like 
short  rows  of 
cocci.  Theyoften 
appear  tapered  at 
one  or  both  ex- 
tremities ;  occa- 
sionally there  is 
slight  club-like 
swelling.  De- 
generated ,  and 
partially  broken 
down  forms  are 
also  seen.  They 
take  up  the  basic 
aniline  stains 
rather  more  read- 

FlG.  97.—  Superficial  part  of  leprous  skin;   the  cells  of  the  ^ 

granulation  tissue  appear  as  dark  patches,  owing  to  the  deeply  bacilli,        but       in 

stained  bacilli  in  their  interior.     In  the  upper  part  a  process  of  j 
epithelium  is  seen. 

Paraffin  section;   stained  with  carbol-fuchsin  and  Bismarck-  them     deeply      a 

brown,     X  500.  r    ,          .    . 

powerful     stain, 

such  as  carbol-fuchsin,  is  necessary.  When  stained,  they  strongly 
resist  decolorising,  though  they  are  more  easily  decolorised  than 
tubercle  bacilli.  The  best  method  is  to  stain  with  carbol-fuchsin 
as  for  tubercle  bacilli,  but  to  use  a  weaker  solution  of  sulphuric 
acid,  say  5  per  cent,  in  decolorising ;  in  the  case  of  films  and  thin 
sections,  decolorising  with  such  a  solution  for  fifteen  seconds  is 
usually  sufficient.  Thereafter  the  tissues  are  coloured  by  a  con- 
trast stain,  such  as  a  watery  solution  of  methylene-blue(W^  p.  104). 
The  bacilli  are  also  readily  stained  by  Gram's  method.  Regard- 
ing the  presence  of  spores  practically  nothing  is  known,  though 
some  of  the  unstained  or  stained  points  may  be  of  this  nature. 
We  have,  however,  no  means  of  testing  their  powers  of  resist- 
ance. Leprosy  bacilli  are  non-motile. 


POSITION    OF   THE   BACILLI.  271 

Position  of  the  Bacilli.  —  They  occur  in  enormous  numbers 
in  the  leprous  lesions,  especially  in  the  tubercular  form.  In 
fact,  so  numer- 
ous are  they 
that  the  granu- 
lation tissue  in 
sections,  prop- 
erly stained  as 
above,  presents 
quite  a  red 
colour  under  a 
low  power  of 
the  microscope. 
The  bacilli  oc- 
cur for  the  most 
part  within  the 
protoplasm  of 
the  round  cells 
of  the  granula- 
tion tissue,  and 

are      often      SO          FIG.  98.  —  High-power  view  of  portion  of  leprous  nodule,  show- 
ing the  arrangement  of  the  bacilli  within  the  cells  of  the  granulation 

numerous  that  tissue. 

the        Structure          Paraffin  section ;  stained  with  carbol-fuchsin  and  methylene-blue. 
X  noo. 

of  the  cells  is 

quite  obscured  (Fig.  97).  They  are  often  arranged  in  bundles 
which  contain  several  bacilli  lying  parallel  to  one  another, 
though  the  bundles  lie  in  various  directions  (Fig.  98).  The 
appearance  thus  presented  by  the  cells  filled  with  bacilli  is 
very  characteristic.  Bacilli  are  also  found  free  in  the  lym- 
phatic spaces,  but  the  greater  number  are  undoubtedly  con- 
tained within  the  cells.  They  are  also  found  in  spindle-shaped 
connective-tissue  cells,  in  endothelial  cells,  and  in  the  walls  of 
blood-vessels.  They  are  for  the  most  part  confined  to  the 
connective  tissue,  but  a  few  may  be  seen  in  the  hair  follicles 
and  glands  of  the  skin.  Occasionally  one  or  two  may  be  found 
in  the  surface  epithelium,  where  they  probably  have  been  carried 
by  leucocytes,  but  this  position  is,  on  the  whole,  exceptional. 
They  also  occur  in  large  numbers  in  the  lymphatic  glands 
associated  with  the  affected  parts.  In  the  internal  organs - 
liver,  spleen,  etc.,  when  leprous  lesions  are  present,  the  bacilli 


2/2  LEPROSY. 

are  also  found,  though  in  relatively  smaller  numbers.  In  the 
nerves  in  the  anaesthetic  form  they  are  comparatively  few,  and 
in  the  sclerosed  parts  it  may  be  impossible  to  find  any.  There 
are  few  also  in  the  skin  patches  referred  to  above  as  occurring 
in  this  form  of  the  disease. 

Their  spread  is  chiefly  by  the  lymphatics,  though  distribution 
by  the  blood  stream  also  occurs.  They  have  been  said  to  be 
found  in  the  blood  during  the  presence  of  fever  and  the  erup- 
tion of  fresh  nodules,  and  they  have  also  been  observed  in  the 
blood-vessels  post  mortem,  being  chiefly  contained  within  leu- 
cocytes. Recent  observations  (e.g.  those  of  Doutrelepont  and 
Wolters)  show  that  the  bacilli  may  be  more  widely  spread 
throughout  the  body  than  was  formerly  supposed.  A  few  may 
be  detected  in  some  cases  in  various  organs  which  show  no 
structural  change,  especially  in  their  capillaries.  The  brain 
and  spinal  cord  are  practically  exempt. 

Relations  to  the  Disease.  —  Attempts  to  cultivate  the  leprosy 
bacilli  outside  the  body  have  so  far  been  unsuccessful.  From 
time  to  time  announcements  of  successful  cultivations  have  been 
made,  but  one  after  another  has  proved  to  be  erroneous.  A 
similar  statement  may  be  made  with  regard  to  experiments  on 
animals.  If  a  piece  of  leprous  tissue  be  introduced  subcuta- 
neously  in  an  animal,  such  as  the  rabbit,  a  certain  amount  of 
induration  may  take  place  around  it,  and  the  bacilli  may  be 
found  unchanged  in  appearance  weeks  or  even  months  after- 
wards, but  no  multiplication  of  the  organisms  occurs.  The 
only  exception  to  this  statement  is  afforded  by  the  experiments 
of  Melcher  and  Orthmann,  who  inoculated  the  anterior  chamber 
of  the  eye  of  rabbits  with  leprous  material,  the  inoculation 
being  followed  by  an  extensive  growth  of  nodules  in  the  lungs 
and  internal  organs,  which  they  affirmed  contained  leprosy 
bacilli.  It  has  been  questioned,  however,  by  several  authorities 
whether  the  organisms  in  the  nodules  were  really  leprosy 
bacilli,  and  up  to  the  present  we  cannot  say  that  there  is  any 
satisfactory  proof  that  the  disease  can  be  transmitted  to  any  of 
the  lower  animals. 

It  would  also  appear  that  the  disease  is  not  readily  inoculable 
in  the  human  subject.  In  a  well-known  case  described  by 
Arning,  a  criminal  in  the  Sandwich  Islands  was  inoculated  in 
several  parts  of  the  body  with  leprosy  tissue.  Two  or  three 


MODE   OF   TRANSMISSION.  2/3 

years  later,  well-marked  tubercular  leprosy  appeared  and  led  to  a 
fatal  result.  This  experiment,  however,  is  open  to  the  objec- 
tion, that  the  individual  before  inoculation  had  been  exposed  to 
infection  in  a  natural  way,  having  been  frequently  in  contact 
with  lepers.  In  other  cases  inoculation  experiments  on  healthy 
subjects,  and  inoculations  in  other  parts  of  leprous  individuals 
have  given  negative  results.  It  has  been  supposed  by  some 
that  the  failure  to  obtain  cultures  and  to  reproduce  the  disease 
experimentally  may  be  partly  due  to  the  bacilli  in  the  tissues 
being  dead.  That  many  of  the  leprou-s  bacilli  are  in  a  dead 
condition  is  quite  possible,  in  view  of  the  long  period  during 
which  dead  tubercle  bacilli  introduced  into  the  tissues  of  ani- 
mals retain  their  form  and  staining  reaction.  There  is  also  the 
fact  that  from  time  to  time  in  leprous  subjects  there  occur 
attacks  of  a  certain  amount  of  fever,  which  are  followed  by  a 
fresh  outbreak  of  nodules,  and  it  would  appear  that  especially 
at  these  times  multiplication  of  the  bacilli  takes  place  more 
actively. 

The  facts  stated  with  regard  to  cultivation  and  inoculation 
experiments  go  to  distinguish  the  leprosy  bacillus  all  the  more 
strongly  from  other  organisms.  Some  have  supposed  that 
leprosy  is  a  form  of  tubercle,  or  tubercle  modified  in  some  way, 
but  for  this  there  appears  to  us  to  be  no  evidence.  Both  from 
the  pathological  and  from  the  bacteriological  point  of  view  the 
diseases  are  distinct.  It  should  also  be  mentioned  that  tubercle 
is  a  not  uncommon  complication  in  leprous  subjects,  in  which 
case  it  presents  the  ordinary  characters. 

The  mode  by  which  leprosy  is  transmitted  has  been  the 
subject  of  great  controversy,  and  is  one  on  which  authorities  still 
hold  opposite  opinions.  Some  consider  that  it  is  a  hereditary 
disease,  or  at  least  that  it  is  transmitted  from  a  parent  to  the 
offspring ;  others  again  that  it  is  transmitted  by  direct  contact. 
There  appears  to  be  no  doubt,  however,  that  on  the  one  hand 
leprous  subjects  may  bear  children  free  from  leprosy,  and 
that,  on  the  other  hand,  healthy  individuals  entering  a  leprous 
district  may  contract  the  disease,  though  this  rarely  occurs. 
Of  the  latter  occurrence  there  is  the  well-known  instance 
of  Father  Damien,  who  contracted  leprosy  after  going  to 
the  Sandwich  Islands.  In  view  of  all  the  facts  there  can  be 
little  doubt  that  leprosy  in  certain  conditions  may  be  transmitted 


2/4  LEPROSY. 

by  direct  contact,  though  its  contagiousness  is  not  of  a  high 
order. 

Methods  of  Diagnosis.  —  Film  preparations  should  be  made 
with  the  discharge  from  any  ulcerated  nodule  which  may  be 
present,  or  from  the  scraping  of  a  portion  of  excised  tissue,  and 
should  be  stained  as  above  described.  The  presence  of  large 
numbers  of  bacilli  situated  within  the  cells  and  giving  the 
staining  reaction  of  leprosy  bacilli,  is  conclusive.  It  is  more 
satisfactory,  however,  to  make  microscopic  sections  through  a 
portion  of  the  excised  tissue,  when  the  structure  of  the  nodule 
and  the  arrangement  of  the  bacilli  can  be  readily  studied.  The 
points  of  difference  between  leprosy  and  tubercle  have  already 
been  stated,  and  in  most  cases  there  is  really  no  difficulty  in 
distinguishing  the  two  conditions. 


CHAPTER   XII. 

GLANDERS  AND   RHINOSCLEROMA. 

GLANDERS. 

THE  bacillus  of  glanders  (bacillus  mallei ;  Fr.  bacille  de  la 
morve  ;  Ger.  Rotzbacillus)  was  discovered  by  Loffler  and  Schiitz, 
the  announcement  of  this  discovery  being  made  towards  the  end 
of  1882.  They  not  only  obtained  pure  cultures  of  this  organism 
from  the  tissues  in  the  disease,  but  by  experiments  on  horses 
and  other  animals  conclusively  established  its  causal  relationship. 
These  have  been  fully  confirmed.  The  same  organism  has  also 
been  cultivated  from  the  disease  in  the  human  subject,  first  by 
Weichselbaum  in  1885,  who  obtained  it  from  the  pustules  in  a 
case  of  acute  glanders  in  a  woman,  and  by  inoculation  of  animals 
obtained  results  similar  to  those  of  Loffler  and  Schiitz. 

Within  recent  years  a  substance,  mallein,  has  been  obtained 
from  the  cultures  of  the  glanders  bacillus  by  a  method  similar  to 
that  by  which  tuberculin  was  prepared,  and  has  been  found  to 
produce  corresponding  effects  in  animals  suffering  from  glanders 
to  those  produced  by  tuberculin  in  tuberculous  animals. 

The  Natural  Disease.  —  Glanders  chiefly  affects  the  equine 
species  —  horses,  mules,  and  asses.  Horned  cattle,  on  the  other 
hand,  are  quite  immune,  whilst  goats  and  sheep  occupy  an 
intermediate  position,  the  former  being  rather  more  susceptible 
and  occasionally  suffering  from  the  natural  disease.  It  also 
occurs  in  some  of  the  carnivora  —  cats,  lions  and  tigers  in 
menageries,  which  animals  are  infected  from  the  carcases  of 
animals  affected  with  the  disease.  Many  of  the  small  rodents 
are  highly  susceptible  to  inoculation  (vide  infra}. 

Glanders  is  also  found  in  man  as  the  result  of  direct  inocula- 
tion on  some  wound  of  the  skin  or  other  part  by  means  of  the 
discharges  or  diseased  tissues  of  an  animal  affected,  and  hence  is 
commonest  amongst  grooms  and  others  whose  work  brings  them 
into  contact  with  horses. 

275 


2/6  GLANDERS   AND   RHINOSCLEROMA. 

In  horses  the  lesions  are  of  two  types,  to  which  the  names  "glanders" 
proper  and  "farcy"  have  been  given,  though  both  may  exist  together.  In 
glanders  proper  the  septum  nasi  and  adjacent  parts  are  chiefly  affected,  there 
occurring  in  the  mucous  membrane  nodules  which  are  at  first  firm  and  of 
somewhat  translucent  grey  appearance.  The  growth  of  these  is  attended 
usually  by  inflammatory  swelling  and  profuse  catarrhal  discharge.  Afterwards 
the  nodules  soften  in  the  centre,  break  down,  and  give  rise  to  irregular  ulcera- 
tions.  Similar  lesions,  though  in  less  degree,  may  be  found  in  the  respiratory 
passages.  Associated  with  these  lesions  there  is  usually  implication  of  the 
lymphatic  glands  in  the  neck,  mediastinum,  etc. ;  and  there  may  be  in  the 
lungs,  spleen,  liver,  etc.,  nodules  of  the  size  of  a  pea  or  larger,  of  greyish  or 
yellow  tint,  firm  or  somewhat  softened  in  the  centre,  and  often  surrounded 
with  a  congested  zone.  The  term  "farcy"  is  applied  to  the  affection  of  the 
superficial  lymphatic  vessels  and  glands,  which  is  specially  seen  where  infection 
takes  place  through  an  abrasion  of  the  skin,  such  as  is  often  produced  by  the 
rubbing  of  the  harness.  The  lymphatic  vessels  become  irregularly  thickened, 
so  as  to  appear  like  knotted  cords,  and  the  associated  lymphatic  glands  be- 
come enlarged  and  firm,  though  suppurative  softening  usually  follows,  and 
there  may  be  ulceration.  These  thickenings  are  often  spoken  of  as  "farcy 
buds-'  and  "farcy  pipes."  In  farcy  also,  secondary  nodules  may  occur  in 
internal  organs  and  the  nasal  mucous  membrane.  In  the  ass  the  disease  runs 
a  more  acute  course  than  in  the  horse. 

In  man  the  disease  is  met  with  in  two  forms,  an  acute  and  a 
chronic  ;  though  intermediate  forms  also  occur,  and  chronic  cases 
may  take  on  the  characters  of  the  acute  disease.  The  site  of 
inoculation  is  usually  on  the  hand  or  arm,  by  means  of  some 
scratch  or  abrasion,  or  possibly  along  a  hair  follicle,  sometimes  on 
the  face,  and  occasionally  on  the  mucous  membrane  of  the  mouth, 
nose,  or  eye.  In  the  acute  form  there  appears  at  the  site  of  in- 
oculation inflammatory  swelling,  attended  usually  with  spreading 
redness,  and  the  lymphatics  in  relation  to  the  part  also  become 
inflamed,  the  appearances  being  those  of  a  "poisoned  wound." 
These  local  changes  are  soon  followed  by  marked  constitutional 
disturbance,  and  by  an  eruption  on  the  surface  of  the  body,  at 
first  papular  and  afterwards  pustular,  and  later  there  may  form  in 
the  subcutaneous  tissue  and  muscles  larger  masses  which  soften 
and  suppurate,  the  pus  being  often  mixed  with  blood  ;  suppuration 
may  occur  also  in  the  joints.  In  some  cases  the  nasal  mucous 
membrane  may  be  secondarily  infected,  and  thence  inflammatory 
swelling  may  spread  to  the  tissues  of  the  face ;  in  others  it 
remains  free.  The  patient  usually  dies  in  two  or  three  weeks, 
sometimes  sooner,  with  the  symptoms  of  rapid  pyaemia.  In 
addition  to  the  lesions  mentioned  there  may  be  foci,  usually 


THE   GLANDERS    BACILLUS.  277 

suppurative,  in  the  lungs  (attended  often  with  pneumonic  con- 
solidation), in  the  spleen,  liver,  bone-marrow,  salivary  glands,  etc. 
In  the  chronic  form  the  local  lesion  results  in  the  formation  of  an 
irregular  ulcer  with  thickened  margins  and  sanious,  often  foul, 
discharge.  The  ulceration  spreads  deeply  as  well  as  superficially, 
and  the  thickened  lymphatics  also  have  a  great  tendency  to 
ulcerate,  though  the  lymphatic  system  is  not  so  prominently 
affected  as  in  the  horse.  Deposits  may  form  in  the  subcutane- 
ous tissue  and  muscles,  and  the  mucous  membrane  may  become 
affected.  The  disease  may  run  a  very  chronic  course,  lasting  for 
months,  and  recovery  may  occur,  though,  on  the  other  hand,  the 
disease  may  take  on  a  more  acute  character  and  rapidly  become 
fatal. 

The  Glanders  Bacillus.  —  Microscopical  Characters,  —  The 
glanders  bacilli  are  minute  rods,  straight  or  slightly  curved,  with 
rounded  ends,  and  about  the 
same  length  as  tubercle  bacilli, 
but  distinctly  thicker  (Fig.  99). 
They  show,  however,  consider- 
able variations  in  size  and  in 
appearance,  and  their  proto- 
plasm is  often  broken  up  into 
a  number  of  deeply-stained  por- 
tions with  unstained  intervals 
between.  These  characters  are 
seen  both  in  the  tissues  and  in 
cultures,  but,  as  in  the  case  of 
many  organisms,  irregularities  FlG.99._GIandersbacimamongstbroken. 

in  form   and   Size  are   more  pro-    down  cells.    Film  preparation  from  a  glanders 

nounced  in  cultures  (Fig.  zoo);  %*£££>?**  stained  with  weak 

short   filamentous   forms   8    to 

12  fji  in  length  are  sometimes  met  with,  but  these  are  on   the 

whole  rare.     The  organism  is  non-motile,  but  brownian  motion  is 

marked. 

In  the  tissues,  the  bacilli  usually  occur  irregularly  scattered 
amongst  the  cellular  elements  ;  a  few  may  be  contained  within 
leucocytes  and  connective-tissue  corpuscles,  but  the  position  of 
most  is  extracellular.  They  are  most  abundant  in  the  acute 
lesions,  in  which  they  may  be  found  in  considerable  numbers ; 
but  in  the  chronic  nodules,  especially  when  softening  has  taken 


278  GLANDERS   AND   RHINOSCLEROMA. 

place,  they  are  few  in  number,  and  it  may  be  impossible  to  find 

any  in  sections.     They  have  less  powers  of  persistence,  and  dis- 

appear in  the  tissues  much  more  quickly,  than  tubercle  bacilli. 

There  has  been  dispute  as  to  whether  or  not  they  contain 

spores.       Some    consider    cer- 

%.  tain  of  the  unstained  portions 

«*  to  be  of  that  nature,  and  it  has 

been  claimed  that  these  can  be 
stained  by  the  method  for  stain- 
ing spores  (Rosenthal).  But  it 
is  very  doubtful  that  such  is  the 
case  ;  the  appearances  corre- 
spond  rather  with  mere  breaks 
in  the  protoplasm,  such  as  are 
,  »%  '  met  with  in  many  other  bacilli 

/  which  do  not  contain   spores, 

and     the     comparatively     low 

FIG.  ioo.  —  Glanders  bacilli  from  a  pure 
culture  on  glycerin  agar.    Stained  with  carbol-   powers  of  resistance  of  glanders 


spores  is  strongly  against  their 
being  of  that  nature.  The  power  of  resistance  is  after  all  the 
important  practical  point. 

Staining.  —  The  glanders  bacillus  differs  widely  from  the 
tubercle  bacillus  in  its  staining  reactions.  It  stains  with  simple 
watery  solutions  of  the  basic  stains,  but  somewhat  faintly  (better 
when  an  alkali  or  a  mordant,  such  as  carbolic  acid,  is  added), 
and  even  when  deeply  stained  it  readily  loses  the  colour  when  a 
decolorising  agent  such  as  alcohol  is  applied.  We  have  obtained 
the  best  results  by  carbol-thionin-blue  (p.  101),  and  we  prefer  to 
dehydrate  by  the  aniline-oil  method.  In  film  preparations  of 
fresh  glanders  nodules  the  bacilli  can  be  readily  found  by  stain- 
ing with  any  of  the  ordinary  combinations,  e.g.  carbol-thionin-blue 
or  weak  carbol-fuchsin.  By  using  a  stain  of  suitable  strength  no 
decolorising  agent  is  necessary,  the  film  being  simply  washed  in 
water,  dried,  and  mounted.  Gram's  method  is  quite  inapplicable, 
the  glanders  bacilli  rapidly  losing  the  stain  in  the  process. 

Cultivation.  —  (For  ttie  methods  of  separation,  vide  infra^ 
The  glanders  bacillus  grows  readily  on  most  of  the  ordinary 
media,  but  a  somewhat  high  temperature  is  necessary,  growth 
taking  place  most  rapidly  at  35°  to  37°  C.  Though  a  certain 


CULTIVATION  OF   BACILLUS   MALLEI.  2/9 

amount  of  growth  occurs  down  to  21°  C.,  a  temperature  above 
25°  C.  is  always  desirable. 

On  agar  and  glycerin  agar  in  stroke-cultures  growth  appears 
along  the  line  as  a  uniform  streak  of  greyish-white  colour  and 
somewhat  transparent  appearance,  with  moist-looking  surface, 
and  when  touched  with  a  needle  is  found  to  be  of  rather  slimy 
consistence.  Later  it  spreads  laterally  for  some  distance,  and 
the  layer  becomes  of  slightly  brownish  tint.  On  serum  the 
growth  is  somewhat  similar  but  more  transparent,  the  separate 
colonies  being  in  the  form  of  round  and  almost  clear  drops.  In 
sub-cultures  on  these  media  at  the  body  temperature  growth  is 
visible  within  twenty-four  hours,  but  when  fresh  cultures  are 
made  from  the  tissues  it  is  not  visible  till  the  second  day. 
Serum,  however,  is  much  more  suitable  for  cultivating  from  the 
tissues  than  the  agar  media;  on  the  latter  it  is  sometimes  difficult 
to  obtain  growth. 

In  broth,  growth  forms  at  first  a  uniform  turbidity,  but  soon 
settles  to  the  bottom,  and  after  a  few  days  forms  a  pretty  thick 
flocculent  deposit  of  slimy  and  somewhat  tenacious  consistence. 

On  potato  at  30°  to  37°  C.  the  glanders  bacillus  flourishes 
well  and  produces  a  characteristic  appearance ;  incubation  at  a 
high  temperature,  however,  being  necessary.  Growth  proceeds 
rapidly,  and  on  the  third  day  has  usually  formed  a  transparent 
layer  of  slightly  yellowish  tint,  like  clear  honey  in  appearance. 
On  subsequent  days,  the  growth  still  extends  and  becomes 
darker  in  colour  and  more  opaque,  till  about  the  eighth  day  it 
has  a  reddish-brown  or  chocolate  tint,  while  the  potato  at  the 
margin  of  the  growth  often  shows  a  greenish-yellow  staining. 
The  characters  of  the  growth  on  potato  along  with  the  micro- 
scopical appearances  are  quite  sufficient  to  distinguish  the  glan- 
ders bacillus  from  every  other  known  organism  (sometimes  the 
cholera  organism  and  the  B.  pyocyaneus  produce  a  somewhat 
similar  appearance,  but  they  can  be  readily  distinguished  by  their 
other  characters).  Potato  is  also  a  suitable  medium  for  starting 
cultures  from  the  tissues ;  in  this  case  minute  transparent  colo- 
nies become  visible  on  the  third  day  and  afterwards  present  the 
appearances  just  described. 

Powers  of  Resistance.  — The  glanders  bacillus  is  not  killed  at 
once  by  drying,  but  usually  loses  its  vitality  after  fourteen  days 
in  the  dry  condition,  though  sometimes  it  lives  longer.  It  is  not 


280  GLANDERS   AND    RHINOSCLEROMA. 

quickly  destroyed  by  putrefaction,  having  been  found  to  be  still 
active  after  remaining  two  or  three  weeks  in  putrefying  fluids. 
In  cultures  the  bacilli  retain  their  vitality  for  three  or  four 
months,  if,  after  growth  has  taken  place,  they  be  kept  at  the 
temperature  of  the  room  ;  on  the  other  hand,  they  are  often 
found  to  be  dead  at  the  end  of  two  weeks  when  kept  constantly 
at  the  body  temperature.  They  have  comparatively  feeble  re- 
sistance to  heat  and  antiseptics.  Loffler  found  that  they  were 
killed  in  ten  minutes  in  a  fluid  kept  at  55°  C.,  and  in  from  two 
to  three  minutes  by  a  5  per  cent  solution  of  carbolic  acid.  Boil- 
ing water  and  the  ordinarily  used  antiseptics  are  very  rapid  and 
efficient  disinfectants. 

We  may  summarise  the  characters  of  the  glanders  bacillus 
by  saying  that  in  its  morphological  characters  it  resembles 
somewhat  the  tubercle  bacillus,  but  is  thicker,  and  differs  widely 
from  it  in  its  staining  reactions.  For  its  cultivation  the  higher 
temperatures  are  necessary,  and  the  growth  on  potato  presents 
most  characteristic  features. 

Experimental  Inoculation.  —  In  horses  subcutaneous  injection 
of  the  glanders  bacillus  in  pure  culture  reproduces  all  the  im- 
portant features  of  the  disease.  This  fact  was  established  at  a 
comparatively  early  date  by  Loffler  and  Schiitz,  who,  after  one 
doubtful  experiment,  successfully  inoculated  two  horses  in  this 
way,  the  cultures  used  having  been  grown  for  several  generations 
outside  the  body.  In  a  few  days  swellings  formed  at  the  sites 
of  inoculation,  and  later  broke  down  into  unhealthy-looking 
ulcers.  One  of  the  animals  died ;  after  a  few  weeks  the  other, 
showing  symptoms  of  cachexia,  was  killed.  In  both  animals,  in 
addition  to  ulcerations  on  the  surface  with  involvement  of  the 
lymphatics,  there  were  found  post  mortem,  nodules  in  the  lungs, 
softened  deposits  in  the  muscles,  and  also  affection  of  the  nasal 
mucous  membrane, — nodules,  and  irregular  ulcerations.  The 
ass  is  even  more  susceptible  than  the  horse,  the  disease  in  the 
former  running  a  more  rapid  course,  but  with  similar  lesions. 
The  ass  can  be  readily  infected  by  simple  scarification  and 
inoculation  with  glanders  secretion,  etc.  (Nocard). 

Of  small  animals,  field-mice  and  guinea-pigs  are  the  most 
susceptible.  Strangely  enough,  house-mice  and  white  mice  en- 
joy  an  almost  complete  immunity.  In  field-mjce  subcutaneous 
inoculation  is  followed  by  a  very  rapid  disease,  usually  leading 


EXPERIMENTAL    INOCULATION.  28 1 

to  death  within  eight  days,  the  organisms  becoming  generalised 
and  producing  numerous  minute  nodules,  especially  in  the  spleen, 
lungs,  and  liver.  In  the  guinea-pig  the  disease  is  less  acute, 
though  secondary  nodules  in  internal  organs  are  usually  present 
in  considerable  numbers.  At  the  site  of  inoculation  an  inflam- 
matory swelling  forms,  which  soon  softens  and  breaks  down, 
leading  to  the  formation  of  an  irregular  crateriform  ulcer  with 
indurated  margins.  The  lymphatic  vessels  become  infiltrated, 
and  the  corresponding  lymphatic  glands  become  enlarged  to  the 
size  of  peas  or  small  nuts,  softened,  and  semi-purulent.  The 
animal  sometimes  dies  in  two  or  three  weeks,  sometimes  not  for 
several  weeks.  Secondary  nodules,  in  varying  numbers  in  dif- 
ferent cases,  may  be  present  in  the  spleen,  lungs,  bones,  nasal 
mucous  membrane,  testicles,  ovaries,  etc.  ;  in  some  cases  a  few 
nodules  are  found  in  the  spleen  alone.  Intraperitoneal  injection 
in  the  male  guinea-pig  is  followed,  as  pointed  out  by  Straus,  by 
a  very  rapid  and  semi-purulent  affection  of  the  tunica  vaginalis, 
shown  during  life  by  great  swelling  and  redness  of  the  testicles, 
which  changes  may  be  noticeable  in  two  or  three  days.  By  this 
method  there  occur  also  numerous  small  nodules  on  the  surface 
of  the  peritoneum.  Rabbits  are  less  susceptible  than  guinea- 
pigs,  and  the  effect  of  subcutaneous  inoculation  is  somewhat 
uncertain.  Accidental  inoculation  of  the  human  subject  with 
pure  cultures  of  the  bacillus  has  in  more  than  one  instance  been 
followed  by  the  acute  form  of  the  disease  and  a  fatal  result. 

Mayer  has  found  that  when  the  glanders  bacillus  is  injected  along  with 
melted  butter  into  the  peritoneum  of  a  guinea-pig,  it  shows  filamentous, 
branching,  and  club-shaped  forms ;  in  other  words,  it  presents  the  characters 
of  a  streptothrix.  Lubarsch,  on  the  other  hand,  in  a  comparative  study  of  the 
results  of  inoculation  with  acid-fast  and  other  bacilli,  found  none  of  the  above 
characters  in  the  case  of  the  glanders  bacillus  (cf.  "  Tubercle  "). 

Action  on  the  Tissues.  —  From  the  above  facts  it  will  be  seen 
that  in  many  respects  glanders  presents  an  analogy  to  tubercle 
as  regards  the  general  characters  of  the  lesions  and  the  mode 
of  spread.  When  the  tissue  changes  in  the  two  diseases  are 
compared,  certain  differences  are  found.  The  glanders  bacillus 
causes  a  more  rapid  and  more  marked  inflammatory  reaction. 
There  is  more  leucocytic  infiltration  and  less  proliferative  change 
which  might  lead  to  the  formation  of  epithelioid  cells.  Thus  the 
centre  of  an  early  glanders  nodule  shows  a  dense  aggregation  of 


282  GLANDERS    AND    RHINOSCLEROMA. 

leucocytes,  many  of  which  are  polymorpho-nuclear,  and  have 
recently  emigrated  from  the  vessels,  whilst  the  tissue  elements 
between  may  be  more  or  less  degenerating,  or  may  show  pro- 
liferative  changes.  And  further,  the  inflammatory  change  may 
be  followed  by  suppurative  softening  of  the  tissue,  especially  in 
certain  situations,  such  as  the  subcutaneous  tissue  and  lymphatic 
glands.  The  nodules,  therefore,  in  glanders,  as  Baumgarten 
puts  it,  occupy  an  intermediate  position  between  miliary  ab- 
scesses and  tubercles.  The  diffuse  coagulative  necrosis  and 
caseation  which  are  so  common  in  tubercle  do  not  occur  to  the 
same  degree  in  glanders,  and  typical  giant-cells  are  not  formed. 
The  nodules  in  the  lungs  show  leucocytic  infiltration  and  thicken- 
ing of  the  alveolar  walls,  whilst  the  vesicles  are  filled  with 
catarrhal  cells ;  i.e.  there  is  reaction  both  on  the  part  of  the 
connective  tissue,  and  of  the  endothelium  of  the  air  vesicles, 
whilst  at  the  periphery  of  the  nodules  connective-tissue  growth 
is  present  in  proportion  to  their  age.  The  tendency  to  spread 
by  the  lymphatics  is  always  a  well-marked  feature,  and  when  the 
bacilli  gain  entrance  to  the  blood  stream,  they  soon  settle  in  the 
various  tissues  and  organs.  Accordingly,  even  in  acute  cases  it 
is  usually  quite  impossible  to  detect  the  bacilli  in  the  circulating 
blood,  though  sometimes  they  have  been  found.  It  is  an  inter- 
esting fact,  shown  by  observations  of  the  disease  both  in  the 
human  subject  and  in  the  horse,  as  well  as  by  experiments  on 
guinea-pigs,  that  the  mucous  membrane  of  the  nose  may  become 
infected  by  means  of  the  blood  stream  —  another  example  of  the 
tendency  of  organisms  to  settle  in  special  sites. 

Mode  of  Spread.  —  Glanders  usually  spreads  from  a  diseased 
animal  by  direct  contagion  with  the  discharge  from  the  nose  or 
from  the  sores,  etc.  So  far  as  infection  of  the  human  subject 
goes,  no  other  mode  is  known.  There  is  no  evidence  that  the 
disease  is  produced  in  man  by  inhalation  of  the  bacilli  in  the 
dried  condition.  Some  authorities  consider  that  pulmonary 
glanders  may  be  produced  in  this  way  in  the  horse,  whilst  others 
maintain  that  in  all  cases  there  is  first  a  lesion  of  the  nasal 
mucous  membrane  or  of  the  skin  surface,  and  that  the  lung  is 
affected  secondarily.  Babes,  however,  found  that  the  disease 
could  be  readily  produced  in  susceptible  animals  by  exposing 
them  to  an  atmosphere  in  which  pulverised  cultures  of  the 
bacillus  had  been.  He  also  found  that  inunction  of  the  skin 


MALLEIN:    ITS   PREPARATION   AND   USE.  283 

with  vaseline  containing  the  bacilli  might  produce  the  disease, 
the  bacilli  in  this  case  entering  along  the  hair  follicles. 

Agglutination  of  Glanders  Bacilli.  —  Shortly  after  the  discovery  of  agglu- 
tination in  typhoid  fever,  M'Fadyean  showed  that  the  serum  of  glandered 
horses  possessed  the  power  of  agglutinating  glanders  bacilli.  His  later 
observations  show  that  in  the  great  majority  of  cases  of  glanders  a  I  in  50 
dilution  of  the  serum  produces  marked  agglutination  in  a  few  minutes,  whilst 
in  the  great  majority  of  non-glandered  animals  no  effect  is  produced  under 
these  conditions.  The  test  performed  in  the  ordinary  way  is,  however,  not 
absolutely  reliable,  as  exceptions  occasionally  occur  in  both  directions,  i.e. 
negative  results  by  glandered  animals  and  positive  results  by  non-glandered 
animals.  He  finds  that  a  more  delicate  and  reliable  method  is  to  grow  the 
bacillus  in  bouillon  containing  a  small  proportion  of  the  serum  to  be  tested. 
In  this  way  he  has  obtained  a  distinct  sedimenting  reaction  with  a  serum 
which  did  not  agglutinate  at  all  distinctly  in  the  ordinary  method.  Further 
observations  are  still  required  to  determine  the  precise  value  of  the  test. 

Mallein  and  its  Preparation.  —  Mallein  is  obtained  from  cultures  of  the 
glanders  bacillus  grown  for  a  suitable  length  of  time,  and,  like  tuberculin,  is 
really  a  mixture  comprising  (i)  substances  in  the  bodies  of  the  bacilli  and  (2) 
their  soluble  products,  not  destroyed  by  heat,  along  with  substances  derived 
from  the  medium  of  growth.  It  was  at  first  obtained  from  cultures  on  solid 
media  by  extracting  with  glycerin  or  water,  but  is  now  usually  prepared  from 
cultures  in  glycerin  bouillon.  Such  a  culture,  after  being  allowed  to  grow  for 
three  or  four  weeks,  is  sterilised  by  heat  either  in  the  autoclave  at  115°  C.  or 
by  steaming  at  100°  C.  on  successive  days.  It  is  then  filtered  through  a 
Chamberland  filter.  The  filtrate  constitutes  fluid  mallein.  Usually  a  little 
carbolic  acid  (.5  per  cent)  is  added  to  prevent  it  from  .-decomposing.  Of  such 
mallein  i  c.c.  is  usually  the  dose  for  a  horse  (M'Fadyean).  Foth  has  pre- 
pared a  dry  form  of  mallein  by  throwing  the  filtrate  of  a  broth  culture,  evapo- 
rated to  one-tenth  of  its  bulk,  into  twenty-five  or  thirty  times  its  volume  of 
alcohol.  A  white  precipitate  is  formed,  which  is  dried  over  calcium  chloride 
and  then  under  an  air-pump.  A  dose  of  this  dry  mallein  is  .05  to  .07  grin, 

The  Use  of  Mallein  as  a  Means  of  Diagnosis.  — In  using  mallein  for  the 
diagnosis  of  glanders,  the  temperature  of  the  animal  ought  to  be  observed  for 
some  hours  beforehand,  and,  after  subcutaneous  injection  of  a  suitable  dose, 
it  is  taken  at  definite  intervals,  —  according  to  M'Fadyean  at  the  sixth,  tenth, 
fourteenth,  and  eighteenth  hours  afterwards,  and  on  the  next  day.  Here  both 
the  local  reaction  and  the  temperature  are  of  importance.  In  a  glandered 
animal,  at  the  site  of  inoculation  there  is  a  somewhat  painful  local  swelling 
which  reaches  a  diameter  of  five  inches  at  least,  the  maximum  size  not  being 
attained  until  twenty-four  hours  afterwards.  The  temperature  rises  1.5°  to 
2°  C.,  or  more,  the  maximum  generally  occurring  in  from  eight  to  sixteen 
hours.  If  the  temperature  never  rises  as  much  as  1.5°,  the  reaction  is  con- 
sidered doubtful.  In  the  negative  reaction  given  by  an  animal  free  from 
glanders,  the  rise  of  temperature  does  not  usually  exceed  i'°,  the  local  swelling 
reaches  the  diameter  of  three  inches  at  most,  and  has  much  diminished  at  the 
end  of  twenty-four  hours.  In  the  case  of  dry  mallein,  local  reaction  is  less 

r^r 

OF  THE 

UNIVERSITY 

OF 


284  GLANDERS   AND    RHINOSCLEROMA. 

marked.  Veterinary  authorities  are  practically  unanimous  as  to  the  great 
value  of  the  mallein  test  as  a  means  of  diagnosis.  We  cannot  as  yet  speak  as 
to  its  applicability  to  diagnosis  of  the  disease  in  the  human  subject. 

Methods  of  Examination  and  Diagnosis.  —  Microscopic  exam- 
ination in  a  case  of  suspected  glanders  will  at  most  reveal  the 
presence  of  bacilli  corresponding  in  their  characters  to  the 
glanders  bacillus.  An  absolute  diagnosis  cannot  be  made  by 
this  method.  Cultures  may  be  obtained  by  making  successive 
strokes  on  blood  serum  or  on  glycerin  agar  (preferably  the 
former),  and  incubating  at  37°  C.  The  colonies  of  the  glanders 
bacillus  do  not  appear  till  two  days  after.  This  method  often 
fails  unless  a  considerable  number  of  the  glanders  bacilli  are 
present.  Another  method  is  to  dilute  the  secretion  or  pus  with 
sterile  water,  to  varying  degrees,  and  then  to  smear  the  surface 
of  potato  with  the  mixture,  the  potatoes  being  incubated  at  the 
above  temperature.  The  colonies  on  potatoes  may  not  appear 
till  the  third  day.  The  most  certain  method,  however,  is  that  of 
Straus  by  inoculation  of  a  male  guinea-pig,  either  by  subcutane- 
ous or  intraperitoneal  injection.  By  the  latter  method,  as  above 
described,  lesions  are  much  more  rapidly  produced,  and  are  more 
characteristic.  If,  however,  there  have  been  other  organisms 
present,  the  animal  may  die  of  a  septic  peritonitis,  though  even  in 
such  a  case  the  glanders  bacilli  will  be  found  to  be  more  numer- 
ous in  the  tunica  vaginalis,  and  may  be  cultivated  from  this 
situation.  Frothingham  recommends  injection  of  the  suspected 
material  into  not  one  guinea-pig,  but  three  or  four,  leaving  a 
small  portion  of  the  injection  beneath  the  skin  ;  by  so  doing 
there  is  less  chance  of  failure  due  either  to  increased  resistance 
on  the  part  of  one  animal,  or  to  lessened  resistance  towards 
the  organisms  in  another  causing  an  early  fatal  peritonitis.  He 
also  warns  one  against  a  negative  diagnosis  being  made  unless 
several  tests  have  been  executed.  In  the  case  of  horses,  etc.,  a 
diagnosis  will,  of  course,  be  much  more  easily  and  rapidly  effected 
by  means  of  mallein. 

RHINOSCLEROMA. 

This  disease  is  considered  here  as,  from  the  anatomical 
changes,  it  also  belongs  to  the  group  of  infective  granulomata. 
It  is  characterised  by  the  occurrence  of  chronic  nodular  thick- 
enings in  the  skin  or  mucous  membrane  of  the  nose,  or  in  the 


BACILLUS   OF   RHINOSCLEROMA.  285 

mucous  membrane  of  the  pharynx,  larynx,  or  upper  part  of  the 
trachea.  It  is  scarcely  ever  met  with  in  this  country,  but  is 
of  not  very  uncommon  occurrence  on  the  Continent,  especially 
in  Austria.  In  the  granulation  tissue  of  the  nodules  there  are 
to  be  found  numerous  round  and  rather  large  cells,  which  have 
peculiar  characters  and  are  often  known  as  the  cells  of  Mikulicz. 
Their  protoplasm  contains  a  collection  of  somewhat  gelatinous 
material  which  may  fill  the  cell  and  push  the  nucleus  to  the  side. 
Within  these  cells  there  is  present  a  characteristic  bacillus, 
occurring  in  little  clumps  or  masses  chiefly  in  the  gelatinous 
material.  A  few  bacilli  also  lie  free  in  the  lymphatic  spaces 
around.  This  organism  was  first  observed  by  Frisch,  and  is  now 
known  as  the  bacillus  of  rhinoscleroma.  The  bacilli  have  the 
form  of  short  oval  rods,  which,  when  lying  separately,  can  be 
seen  to  possess  a  distinct  capsule,  and  which  in  all  their  micro- 
scopical characters  correspond  closely  with  Friedlander's  pneumo- 
bacillus.  They  are  usually  present  in  the  lesions  in  a  state  of 
purity.  It  was  at  first  stated  that  they  could  be  stained  by 
Gram's  method,  but  more  recent  observations  show  that  like 
Friedlander's  organism  they  lose  the  stain. 

From  the  affected  tissues  this  bacillus  can  be  easily  cultivated 
by  the  ordinary  methods.  In  the  characters  of  its  growth  in  the 
various  culture  media  it  presents  a  close  similarity  to  that  of  the 
pneumobacillus,  as  it  also  does  in  its  fermentative  action  in  milk 
and  sugar-containing  fluids.  The  nail-like  appearance  of  the 
growth  on  gelatin  is  said  to  be  less  distinct,  and  the  growth  on 
potatoes  is  more  transparent  and  may  show  small  bubbles  of 
gas ;  otherwise  it  resembles  the  pneumobacillus.  It  is  doubtful 
whether  any  distinct  line  of  difference  can  be  drawn  between 
the  two  organisms  so  far  as  their  microscopical  and  cultural 
characters  are  concerned. 

The  evidence  that  the  organisms  described  are  the  cause  of 
this  disease  consists  in  their  constant  presence  and  their  special 
relation  to  the  affected  tissues,  as  already  described.  From  these 
facts  alone  it  is  highly  probable  that  they  are  the  active  agents 
in  the  production  of  the  lesions.  Experimental  inoculation  has 
thrown  little  light  on  the  subject,  though  one  observer  has 
described  the  production  of  nodules  on  the  conjunctivae  of 
guinea-pigs.  The  relation  of  the  rhinoscleroma  organism  to 
that  of  Friedlander  is,  however,  still  a  matter  of  doubt,  and  the 


286  GLANDERS   AND    RHINOSCLEROMA. 

matter  has  been  further  complicated  by  the  fact  that  a  bacillus 
possessing  closely  similar  characters  has  been  found  to  be  very 
frequently  present  in  ozcena,  and  is  often  known  as  the  bacillus 
ozoence.  The  last-mentioned  organism  is  said  to  have  more  active 
fermentative  powers.  From  what  has  been  stated  it  will  be 
seen  that  a  number  of  organisms,  closely  allied  in  their  morpho- 
logical characters,  have  been  found  in  the  nasal  cavity  in  healthy 
or  diseased  conditions.  From  what  we  know,  however,  of  other 
diseases,  it  is  not  improbable  that  though  presenting  these  close 
resemblances,  they  may  be  distinct  species,  and  may  cause 
distinct  pathological  conditions  in  man.  The  subject  is  one  on 
which  more  light  is  still  required. 


CHAPTER   XIII. 
ACTINOMYCOSIS   AND  ALLIED   DISEASES. 

ACTINOMYCOSIS  is  a  disease  of  special  interest,  inasmuch  as 
it  is  the  most  important  example  of  a  microbic  affection  in  which 
the  parasite  belongs  to  the  higher  order  of  bacteria.  It  is 
related,  by  the  characters  of  the  pathological  changes,  to  the 
diseases  which  have  been  described. 

The  disease  affects  man  in  common  with  certain  of  the 
domestic  animals,  though  it  is  more  frequent  in  the  latter, 
especially  in  oxen,  swine,  and  horses.  The  parasite  was  first 
discovered  in  the  ox  by  Bollinger,  and  described  by  him  in 
1877,  the  name  actinomyces  or  ray  fungus  being  from  its  appear- 
ance applied  to  it  by  the  botanist  Harz.  In  1878  Israel  de- 
scribed the  parasite  in  the  human  subject,  and  in  the  following 
year  Ponfick  identified  it  as  being  the  same  as  that  found  in  the 
ox.  Since  that  time  a  large  number  of  cases  have  been  observed 
in  the  human  subject,  the  result  of  investigation  being  to  show 
that  it  affects  man  much  more  frequently  than  was  formerly 
supposed. 

It  is,  however,  very  probable  that  the  term  actinomyces  does 
ndt  represent  one  parasite  but  a  number  of  closely  allied  spe- 
cies, as  cultures  obtained  from  various  sources  have  presented 
considerable  differences.  It  is  also  to  be  noted  that  other  dis- 
tinct species  of  streptothrix l  have  been  cultivated  from  isolated 
cases  of  disease  in  the  human  subject  where  the  lesions  resem- 
bled more  or  less  closely  those  of  actinomycosis.  In  one  or 
two  instances  the  organism  has  been  found  to  be  "  acid-fast," 
and  there  is  no  doubt  that  the  actinomyces  group  is  closely 
related  through  intermediate  forms  with  the  tubercle  group 
(vide  p.  240). 

Naked-eye  Characters  of  the  Parasite.  —  The  actinomyces 
grows  in  the  tissues  in  the  form  of  little  round  masses  or  colo- 
nies, which,  when  fully  developed,  are  easily  visible  to  the  naked 

1  See  footnote  to  p.  16. 
287 


288  ACTINOMYCOSIS  AND    ALLIED   DISEASES. 

eye,  the  largest  being  about  the  size  of  a  small  pin's  head,  whilst 
all  sizes  below  this  may  be  found.  When  suppuration  is  pres- 
ent, they  lie  free  in  the  pus ;  when  there  is  no  suppuration,  they 
are  embedded  in  the  granulation  tissue,  but  are  usually  sur- 
rounded by  a  zone  of  softer  tissue.  They  may  be  transparent 
or  jelly-like,  or  they  may  be  opaque  and  of  various  colours  — 
white,  yellow,  greenish,  or  almost  black.  The  appearance  de- 
pends upon  their  age  and  also  upon  their  structure,  the  younger 
colonies  being  more  or  less  transparent,  the  older  ones  being  gen- 
erally opaque.  Their  colour  is  modified  by  the  presence  of  pig- 
ment and  by  degenerative  change,  which  is  usually  accompanied 
by  a  yellowish  coloration.  They  are  generally  of  soft,  some- 
times tallow-like,  consistence,  though  sometimes  in  the  ox  they 
are  gritty,  owing  to  the  presence  of  calcareous  deposit.  They 
may  be  readily  found  in  the  pus  by  spreading  it  out  in  a  thin 
layer  on  a  glass  slide  and  holding  it  up  to  the  light.  They  are 
sometimes  described  as  being  always  of  a  distinctly  yellow 
colour,  but  this  is  only  occasionally  the  case ;  in  fact,  in  the 
human  subject  they  occur  much  more  frequently  as  small 
specks  of  semi-translucent  appearance,  and  of  greenish-grey 
tint. 

Microscopical  Characters.  —  The  parasite,  which  is  now  gen- 
erally regarded  as  belonging  to  the  streptothrix  group  of  the 
higher  bacteria  (the  actinomycetes  group  of  Lachner-Sandoval, 
p.  16),  presents  pleomorphous  characters.  In  the  colonies,  as 
they  grow  in  the  tissues,  three  morphological  elements  may  be 
described,  namely,  filaments,  coccus-like  bodies,  and  clubs. 

i.  The  filaments  are  comparatively  thin,  measuring  about 
.5  At  in  diameter,  but  they  are  often  of  great  length.  They  are 
composed  of  a  central  protoplasm  enclosed  by  a  sheath.  The 
latter,  which  is  most  easily  made  out  in  the  older  filaments  with 
granular  protoplasm,  occasionally  contains  granules  of  dark 
pigment.  In  the  centre  of  the  colony  the  filaments  interlace 
with  one  another,  and  form  an  irregular  network  which  may  be 
loose  or  dense ;  at  the  periphery  they  are  often  arranged  in  a 
somewhat  radiating  manner,  and  run  outwards  in  a  wavy  or  even 
spiral  course.  They  also  show  branching,  a  character  which  at 
once  distinguishes  them  from  the  ordinary  bacteria.  Between 
the  filaments  there  is  a  finely  granular  or  homogeneous  ground 
substance.  Most  of  the  colonies  at  an  early  stage  are  chiefly 


MICROSCOPICAL  CHARACTERS. 


289 


constituted  by  filaments  loosely  arranged ;  but  later,  part  of  the 
growth  may  become  so  dense  that  its  structure  cannot  be  made 
out.  This  dense  part,  starting  excentrically,  may  grow  round 
the  colony  to  form  a  hollow  sphere,  from  the  outer  surface  of 
which  filaments  radiate  for  a  short  distance  (Fig.  101).  The 
filaments  usually  stain  uniformly  in  the  younger  colonies,  but 
some,  especially  in  the  older  colonies,  may  be  segmented  so  as 
to  give  the  appearance  of  a  chain  of  bacilli  or  of  cocci,  though 


FlG.  101.  —  Actinomycosis  of  human  liver,  showing  a  colony  of  the  parasite  composed 
of  a  felted  mass  of  filaments  surrounded  by  pus. 

Paraffin  section  ;  stained  by  Gram's  method  and  with  safranin.     X  500. 

the  sheath  enclosing  them  may  generally  be  distinguished. 
Rod-shaped  and  spherical  forms  may  also  be  seen  lying  free. 
2.  Coccus-like  Bodies.  —  The  formation  of  these  from  fila- 
ments has  already  been  described,  but  it  is  doubtful  if  all  are  of 
the  same  nature.  Like  other  species  of  streptothrix,  the  acti- 
nomyces  when  growing  on  a  culture  medium  shows  on  its  sur- 
face filaments  growing  upwards  in  the  air,  the  protoplasm  of 
which  becomes  segmented  into  rounded  spores  or  gonidia.  In 
natural  conditions  outside  the  body  these  gonidia  become  free 
and  act  as  new  centres  by  growing  out  into  filaments.  They 
have  somewhat  higher  powers  of  resistance  than  the  filaments, 
though  less  than  the  spores  of  most  of  the  lower  bacteria.  It  is 


290 


ACTINOMYCOSIS   AND   ALLIED   DISEASES. 


probable  that  some  of  the  spherical  bodies  formed  within  fila- 
ments when  growing  in  the  tissues  have  the  same  significance, 
i.e.  are  gonidia,  whilst  others  may  be  merely  the  result  of  de- 
generative change.  Some  observers  have  described  young 
colonies  largely  composed  of  spherical  forms,  as  if  these  multi- 
plied by  division,  but  this  latter  point  is  still  doubtful.  Both 
the  filaments  and  the  spherical  bodies  are  readily  stained  by 
Gram's  method. 


<"+ 


« • » •  ft 


»X^j*~:  .Y^ 

,\*-'sv4s  s  iv-*  :•••#•. 

-•r^r^^iifi'  & 


FlG.  102.  —  Actinomyces  in  human  kidney,  showing  clubs  radially  arranged  and  sur- 
rounded by  pus.  The  filaments  had  practically  disappeared. 

Paraffin  section  ;  stained  with  haematoxylin  and  rubin.     X  500. 

3.  Clubs.  —  These  are  elongated  pear-shaped  bodies  which 
are  seen  at  the  periphery  of  the  colony,  and  are  formed  by  a 
sort  of  hyaline  swelling  of  the  sheath  around  the  free  extremity 
of  a  filament  (Figs.  102,  103).  They  are  usually  homogeneous 
and  structureless  in  appearance.  In  the  human  subject  the 
clubs  are  often  comparatively  fragile  structures  which  are  easily 
broken  down  and  may  sometimes  be  dissolved  in  water.  Some- 
times they  are  well  seen  when  examined  in  the  fresh  condition, 
but  in  hardened  specimens  are  no  longer  distinguishable.  In 
specimens  stained  by  Gram's  method  they  are  not  coloured  by 
the  violet,  but  take  readily  a  contrast  stain,  such  as  picric  acid, 
rubin,  etc. ;  sometimes  a  darkly-stained  filajnent  can  be  seen 


MICROSCOPICAL  CHARACTERS. 


291 


running  for  a  distance  in  the  centre,  and  may  have  a  knob-like 
extremity.  In  many  of  the  colonies  in  the  human  subject  the 
clubs  are  absent.  In  the  ox,  on  the  other  hand,  where  there  are 
much  older  colonies,  the  clubs  constitute  the  most  prominent 
feature,  whilst  in  most  colonies  the  filaments  are  more  or  less 
degenerated,  and  it  may  sometimes  be  impossible  to  find  any. 
They  often  form  a  dense  fringe  around  the  colony,  and  when: 
stained  by  Gram's  method  retain  the  violet  stain.  They  have,, 
in  fact,  undergone  some  further  chemical  change  which  produces 


*^ 

FIG.  103.  —  Colonies  of  actinomycosis,  showing  general  structural  arrangement  and 
clubs  at  periphery.     From  pus  in  human  subject. 
Stained  by  Gram  and  safranin.     x  60. 

the  altered  staining  reaction.  Clubs  showing  intermediate  stain- 
ing reaction  have  been  described  by  M'Fadyean.  The  view 
that  the  clubs  are  organs  of  fructification  has  been  abandoned 
by  most  authorities,  and  there  appears  to  us  little  evidence  in 
support  of  it. 

Tissue  Lesions.  —  In  the  human  subject  the  parasite  produces 
by  its  growth  a  chronic  inflammatory  change,  usually  ending  in 
a  suppuration  which  slowly  spreads.  In  some  cases  there  is  a 
comparatively  large  production  of  granulation  tissue,  with  only  a 
little  softening  in  the  centre,  so  that  the  mass  feels  solid.  This 


292  ACTINOMYCOSIS    AND   ALLIED   DISEASES. 

condition  is  sometimes  found  in  the  subcutaneous  tissue,  espe- 
cially when  the  disease  has  not  advanced  far,  and  also  in  dense 
fibrous  tissue.  In  most  cases,  however,  and  especially  in  inter- 
nal organs,  suppuration  is  the  outstanding  feature.  This  is  to 
be  associated  with  abundant  growth  of  the  parasite  in  the  fila- 
mentous form.  In  an  organ  such  as  the  liver,  multiple  foci  of 
suppuration  are  seen  at  the  spreading  margin  of  the  disease, 
presenting  a  honeycomb  appearance  which  is  somewhat  charac- 
teristic, whilst  the  colonies  of  the  parasite  may  be  seen  in  the 
pus  with  the  naked  eye.  In  the  older  parts  the  abscesses  have 
become  confluent,  and  formed  large  areas  of  suppuration.  The 
pus  is  usually  of  greenish-yellow  colour  and  of  somewhat  slimy 
character. 

In  cattle  the  tissue  reaction  is  more  of  a  formative  type,  there 
being  abundant  growth  of  granulation  tissue,  which  may  result 
in  large  tumour-like  masses,  usually  of  more  or  less  nodulated 
character,  and  often  consisting  of  well-developed  fibrous  tissue 
containing  areas  of  younger  formation  in  which  irregular  abscess 
formation  is  usually  present.  The  cells  immediately  around  the 
colonies  are  usually  irregularly  rounded,  or  may  even  be  some- 
what columnar  in  shape,  whilst  farther  out  they  become  spindle- 
shaped  and  concentrically  arranged.  It  is  not  uncommon  to 
find  leucocytes  or  granulation  tissue  invading  the  substance  of 
the  colonies,  and  portions  of  the  parasite,  etc.,  may  be  contained 
within  leucocytes  or  within  small  giant-cells  which  are  sometimes 
present.  A  similar  invasion  of  old  colonies  by  leucocytes  is 
sometimes  seen  in  human  actinomycosis. 

Origin  and  Distribution  of  Lesions. — The  lesions  in  the  human 
subject  may  occur  in  almost  any  part  of  the  body,  the  paths  of 
entrance  being  very  various.  In  many  cases  the  entrance  takes 
place  in  the  region  of  the  mouth  —  probably  around  a  decayed 
tooth  —  by  the  crypts  of  the  tonsil,  or  by  some  abrasion.  Swell- 
ing and  suppuration  may  then  follow  in  the  vicinity  and  may 
spread  in  various  directions.  The  periosteum  of  the  jaw  or  the 
vertebrae  may  thus  become  affected,  caries  or  necrosis  resulting, 
or  the  pus  may  spread  deeply  in  the  tissues  of  the  neck,  and 
may  even  pass  into  the  mediastinum.  Occasionally  the  parasite 
may  enter  the  tissues  from  the  oesophagus,  and  in  a  considerable 
number  of  cases  the  primary  lesion  is  in  some  part  of  the  intes- 
tine, generally  of  the  large  intestine.  The  parasite  penetrates 


DISTRIBUTION    OF   THE  LESIONS. 


293 


the  wall  of  the  bowel,  and  may  be  found  deeply  between  the 
coats,  surrounded  by  purulent  material.  Ulceration,  and  some- 
times a  considerable  amount  of  necrosis,  may  follow.  Thence  it 
may  spread  to  the  peritoneum  or  to  the  extraperitoneal  tissue, 
the  retrocaecal  connective  tissue  and  that  around  the  rectum 
being  not  uncommonly  seats  of  suppuration  produced  in  this 
way.  A  peculiar  affection  of  the  intestine  has  been  described, 
in  which  slightly  raised  placques  are  found  both  in  the  large 
and  small  intestines,  these  placques  being  composed  almost  ex- 
clusively of  masses  of  the  actinomyces  along  with  epithelial 
cells.  This,  however,  is  a  very  rare  condition.  The  path  of 
entrance  may  also  be  by  the  respiratory  passages,  the  primary 
lesion  being  pulmonary  or  peribronchial ;  extensive  suppuration 
in  the  lungs  may  result.  Infection  may  also  occur  by  the  skin 
surface,  and,  lastly,  by  the  female  genital  tract,  "as  in  a  case 
recorded  by  Grainger  Stewart  and  Muir,  in  which  both  ovaries 
and  both  Fallopian  tubes  were  affected. 

When  the  parasite  has  invaded  the  tissues  by  any  of  these 
channels,  secondary  or  "  metastatic  "  abscesses  may  occur  in  in- 
ternal organs.  The  liver  is  the  organ  most  frequently  affected, 
though  abscesses  may  occur  in  the  lungs,  brain,  kidneys,  etc. 
In  such  cases  the  spread  takes  place  by  the  blood  stream,  and 
it  is  possible  that  leucocytes  may  be  the  carriers  of  the  infection, 
as  it  is  not  uncommon  to  find  leucocytes  in  the  neighbourhood 
of  a  colony  containing  small  portions  of  the  filaments  in  their 
interior. 

In  the  ox,  on  the  other  hand,  the  disease  usually  remains 
quite  local,  or  spreads  by  continuity.  It  may  produce  tumour- 
like  masses  in  the  region  of  the  jaw  or  neck,  or  it  may  specially 
affect  the  palate  or  tongue,  in  the  latter  producing  enlargement 
and  induration,  with  nodular  thickening  on  the  surface — the 
condition  known  as  "woody  tongue." 

Source  of  the  Parasite.  —  There  is  a  considerable  amount  of 
evidence  to  show  that  outside  the  body  the  parasite  grows  on 
grain,  especially  on  barley.  Both  in  the  ox  and  in  the  pig  the 
parasite  has  been  found  growing  around  fragments  of  grain 
embedded  in  the  tissues.  There  are  besides,  in  the  case  of  the 
human  subject,  a  certain  number  of  cases  in  which  there  was  a 
history  of  penetration  of  a  mucous  surface  by  a  portion  of  grain, 
and  in  a  considerable  proportion  of  cases  the  patient  has  been 


294 


ACTINOMYCOSIS    AND   ALLIED   DISEASES. 


exposed  to  infection  from  this  source.     The  position  of   the 
lesions  in  cattle  is  also  in  favour  of  such  a  view. 

Cultivation  (for  methods  of 
isolation  see  later). — The  actino- 
myces  grows  on  a  variety  of 
media,  though  on  all  its  rate 
of  growth  is  somewhat  slow. 
Growth  takes  place  at  the  ordi- 
nary room  temperature,  but  very 
slowly,  the  temperature  of  the 
body  being  much  more  suitable, 
and  it  would  seem  that  an  ana- 
erobic condition  is  more  produc- 
tive of  positive  results  in  many 
instances  than  where  oxygen  is 
not  excluded. 

On  agar  or  glycerin  agar  at 
37°  C,  growth  is  generally  visi- 
ble on  the  third  or  fourth  day  in 
the   form    of    little   transparent 
FiG.io4.-Cuituresoftheactinomyceson  drops  which  gradually  enlarge 

glycerin  agar,  of  about  three  weeks' growth,    and  form  rounded  projections  of 
showing    the    appearances    which    occur. 

The  growth  in  A  is  at  places  somewhat  a  reddish-yellow  tint  and  some- 

corrugated  on  the  surface.     Natural  size.        what      transparent     appearance, 

like  drops  of  amber.  The  growths  tend  to  remain  separate,  and 
even  when  they  become  con- 
fluent, the  nodular  character 
is  maintained.  They  have  a 
tough  consistence,  being  with 
difficulty  broken  up,  and  ad- 
here firmly  to  the  surface  of 
the  agar.  Older  growths  often 
show  on  the  surface  a  sort  of 
corrugated  aspect,  and  may 
sometimes  present  the  appear- 
ance of  having  been  dusted 
with  a  brownish-yellow  powder 
(Fig.  104).  The  organism 
grows  well  in  the  anaerobic 

Condition  On  agar,  and  for  this    filaments.     Stained  with  fuchsin. 


VARIETIES   OF   ACTINOMYCES. 


295 


purpose  unopened  eggs  also,  either  in  the  fresh  or  boiled  con- 
dition, have  been  used,  inoculation  being  effected  by  drilling  in 
the  shell  a  small  hole  which  is  afterwards  closed.  The  growth 
on  potatoes  is  somewhat  similar  to  that  on  agar. 

On  gelatin  the  same  tendency  to  grow  in  little  spherical 
masses  is  seen,  and  the  medium  becomes  very  slowly  liquefied. 
When  this  occurs  the  liquefied  portion  has  a  brownish  colour 
and  somewhat  syrupy  consistence,  and  the  growths  may  be  seen 
at  the  bottom,  as  little  balls,  from  the  surface  of  which  filaments 
radiate. 

In  the  cultures  at  an  early  stage  the  growth  is  composed  of 
branching  filaments,  which  stain  uniformly  (Fig.  105),  but  later 
some  of  the  superficial  filaments  may  show  segmentation  into 
gonidia.  True  clubs  are  not  formed  in  cultures,  though  slight 
bulbous  thickenings  may  be  seen  at  the  end  of  some  of  the 
filaments. 

Varieties  of  Actinomyces  and  Allied  Forms.  —  It  is  probable  that  in  the 
cases  of  the  disease  described  in  the  human  subject  there  are  more  than 
one  variety  or  species  of  parasite  belonging  to  the  same  group.  Gasperini 
has  described  several  varieties  of  actinomyces  boms  according  to  the  colour  of 
the  growths,  and  a  similar  condition  may  obtain  in  the  case  of  the  human 
subject.  Furthermore  a  considerable  number  of  sir eptothr ices  have  been  found 
in  cases  of  disease  in  the  human  subject,  the  associated  lesions  varying  in 
character  from  tubercle-like  nodules  on  the  one  hand  to  suppurative  processes 
on  the  other.  The  organisms  cultivated  from  such  sources  differ  according  to 
their  microscopic  characters  (for  example,  some  form  u  clubs,"  whilst  others 
do  not),  according  to  their  conditions  of  growth,  staining  reactions,  etc.  Of 
these  only  a  few  examples  may  here  be  mentioned,  but  it  may  be  noted  that 
the  importance  of  the  streptothrices  as  causes  of  disease  is  constantly  being 
extended.  Wolff  and  Israel  cultivated  from  two  cases  of  "  actinomycosis  "  in 
man  a  streptothrix  which  differs  in  so  many  important  points  from  the  actino- 
myces that  it  is  now  regarded  as  a  distinct  species.  Another  species  was 
cultivated  by  Eppinger  from  a  brain  abscess,  and  called  by  him  "  cladothrix 
asteroides,"  from  the  appearance  of  its  colonies  on  culture  media.  In  the 
tissues  it  grows  in  a  somewhat  diffuse  manner  and  does  not  form  clubs  ;  in 
rabbits  and  guinea-pigs  it  produces  tubercle-like  lesions.  Flexner  observed 
a  streptothrix  in  the  lungs  associated  with  lesions  somewhat  like  a  rapid 
phthisis,  and  applied  the  name  "  pseudo-tuberculosis  hominis  streptothricea"  ; 
an  apparently  similar  condition  has  been  described  by  Buchholz.  Berestnew 
cultivated  two  species  of  streptothrix^from  suppurative  lesions,  one  of  which  is 
acid-fast  and  grows  only  in  anaerobic  conditions.  Quite  recently  Birt  and 
Leishman  have  described  another  acid-fast  streptothrix  obtained  from  cirrhotic 
nodules  in  the  lungs  of  a  man.  This  organism  grows  readily  on  ordinary 
media,  forming  a  white  powdery  growth  which  afterwards  assumes  a  pinkish 


296  ACTINOMYCOSIS   AND    ALLIED   DISEASES. 

colour ;  it  is  pathogenic  for  guinea-pigs,  in  which  it  causes  caseous  lesions. 
There  is,  further,  the  streptothrix  madurae  described  below. 

In  diseases  of  the  lower  animals  several  other  forms  have  been  found. 
For  example,  a  streptothrix  has  been  shown  by  Nocard  to  be  the  cause  of  a 
disease  of  the  ox,  —  "farcin  du  boeuf," —  a  disease  in  which  also  there  occur 
tumour-like  masses  of  granulation  tissue.  Dean  has  cultivated  from  a  nodule 
in  a  horse  another  streptothrix,  which  produces  tubercle-like  nodules  in  the 
rabbit,  with  club  formation ;  it  has  close  resemblances  to  the  organism  of  Israel 
and  Wolff.  The  so-called  diphtheria  of  calves  and  "  bacillary  necrosis  "  in 
the  ox  are  probably  both  produced  by  another  streptothrix  or  leptothrix 
which  grows  diffusely  in  the  tissues  in  the  form  of  fine  felted  filaments.  Fur- 
ther investigation  may  show  that  some  of  these  or  other  species  may  occur  in 
the  human  subject  in  conditions  which  are  not  yet  differentiated. 

Experimental  Inoculation.  —  Inoculation  of  smaller  animals, 
such  as  rabbits  and  guinea-pigs,  has  usually  failed  to  give  posi- 
tive results.  This  was  the  case,  for  example,  in  the  important 
series  of  experiments  by  Bostrom,  and  it  may  be  assumed  that 
these  animals  are  little  susceptible  to  the  actinomyces.  The 
disease  has,  however,  been  experimentally  produced  in  the 
bovine  species  both  by  cultures  from  the  ox  and  from  the 
human  subject.  Inoculation  with  the  streptothrix  of  Israel  and 
Wolff  produces  nodular  lesions  both  in  rabbits  and  in  guinea- 
pigs,  and  a  similar  result  has  been  obtained  with  several  of  the 
other  species  of  streptothrix  mentioned  above. 

Methods  of  Examination  and  Diagnosis.  —  As  actinomycosis 
cannot  be  diagnosed  with  certainty  apart  from  the  discovery  of 
the  parasite,  a  careful  examination  of  the  pus  in  obscure  cases 
of  suppuration  should  always  be  undertaken.  As  already  stated, 
the  colonies  can  be  recognised  with  the  naked  eye,  especially 
when  some  of  the  pus  is  spread  out  on  a  piece  of  glass.  If  one 
of  these  is  washed  in  salt  solution  and  examined  unstained,  the 
clubs,  if  present,  are  at  once  seen  on  microscopic  examination. 
Or  the  colony  may  be  stained  with  a  simple  reagent  such  as 
picrocarmine,  and  mounted  in  glycerin  or  Farrant's  solution. 
To  study  the  filaments,  a  colony  should  be  broken  down  on  a 
cover-glass,  dried,  and  stained  with  a  simple  solution  of  any  of 
the  basic  aniline  dyes,  such  as  gentian-violet,  though  better 
results  are  obtained  by  carbol-thionin-blue,  or  by  carbol-fuchsin 
diluted  with  five  parts  of  water.  If  the  specimen  be  overstained, 
it  may  be  decolorised  by  weak  acetic  acid.  Cover-glass  prepa- 
rations of  this  kind  and  also  of  cultures  are  readily  stained  by 


MADURA  DISEASE.  297 

these  methods,  but  in  the  case  of  sections  of  the  tissues  Gram's 
method,  or  a  modification  of  it,  should  be  used  to  show  the 
filaments,  etc.,  a  watery  solution  of  rubin  being  afterwards  used 
to  stain  the  clubs.  By  this  method,  very  striking  preparations 
may  be  obtained. 

To  obtain  cultures,  tubes  of  one  per  cent  glucose  broth 
whose  reaction  is  1.5  per  cent  acid  to  phenol-phthaleine,  and  tubes 
of  glycerin  agar  of  similar  reaction,  should  be  inoculated  with 
portions  of  the  colonies  and  incubated,  anaerobically  as  well  as 
aerobically,  at  37°  C.  Owing  to  the  slow  growth  of  the  actino- 
myces,  however,  the  obtaining  of  pure  cultures  is  difficult,  unless 
the  pus  is  free  from  contamination  with  other  organisms. 

MADURA  DISEASE. 

Madura  disease,  or  mycetoma,  resembles  actinomycosis  both 
as  regards  the  general  characters  of  the  lesions  and  the  occur- 
rence of  the  parasite  in  the  form  of  colonies  or  "granules." 
There  is  no  doubt,  however,  that  the  two  conditions  are  dis- 
tinct, and  it  also  appears  established  that  the  two  varieties  of 
Madura  disease  (vide  infra)  are  produced  by  different  organisms. 
This  disease  is  comparatively  common  in  India  and  in  various 
other  parts  of  the  tropics :  it  has  also  been  met  with  in  Algiers 
and  in  America.  Madura  disease  differs  from  actinomyces  not 
only  in  its  geographical  distribution,  but  also  in  its  clinical  char- 
acters. Its  course,  for  example,  is  of  an  extremely  chronic 
nature,  and  though  the  local  disease  is  incurable  except  by 
operation,  the  parasite  never  produces  secondary  lesions  in 
internal  organs.  Vincent  also  found  that  iodide  of  potassium, 
which  has  a  high  value  as  a  therapeutic  agent  in  many  cases 
of  actinomycosis,  had  no  effect  in  the  case  of  Madura  disease 
studied  by  him.  It  most  frequently  affects  the  foot;  hence  the 
disease  is  often  spoken  of  as  "  Madura  foot."  The  hand  is 
rarely  affected.  In  the  parts  affected  there  is  a  slow  growth 
of  granulation  tissue  which  has  an  irregularly  nodular  character, 
and  in  the  centre  of  the  nodules  there  occurs  purulent  softening 
which  is  often  followed  by  the  formation  of  fistulous  openings 
and  ulcers.  There  are  great  enlargement  and  distortion  of  the 
part  and  frequently  caries  and  necrosis  of  the  bones.  Within 
the  softened  cavities  and  also  in  the  spaces  between  the  fibrous 
tissue,  small  rounded  bodies  or  granules,  bearing  a  certain 


298 


ACTINOMYCOSIS   AND   ALLIED   DISEASES. 


resemblance  to  the  actinomyces,  are  present.  These  may  have 
a  yellowish  or  pinkish  colour,  compared  from  their  appearance 
to  fish  roe,  or  they  may  be  black  like  grains  of  gunpowder,  and 
may  by  their  conglomeration  form  nodules  of  considerable  size. 
Hence  a  pale  variety  and  a  black  variety  of  the  disease  have 
been  distinguished;  in  both  varieties  the  granules  mentioned 
reach  a  rather  larger  size  than  in  actinomycosis.  These  two 
conditions  will  be  considered  separately. 

Pale  Variety.  — When  the  roe-like  granules  are  examined 
microscopically,  they  are  found,  like  the  actinomyces,  to  show 
in  their  interior  an  abundant  mass  of  branching  filaments  with 
mycelial  arrangement  (Fig.  106).  There  may  also  be  present  at 

the  periphery  club-like  struc- 
tures, as  in  actinomyces;  some- 
times they  are  absent.  These 
structures  often  have  an  elon- 
gated wedge  shape,  forming  an 
outer  zone  to  the  colony,  and 
in  some  cases  the  filaments  can 
be  found  to  be  connected  with 
them.  Vincent  obtained  cul- 
tures of  the  parasite  from  a 
*  y/  case  in  Algiers,  and  found  it 
^v^^  f*.^  to  be  a  distinct  species :  it  is 

now  known  as  the  streptothrix 
maditra.  Morphologically  it 
closely  resembles  the  actino- 
myces, but  it  presents  certain 

differences  in  cultural  characters.  In  gelatin  it  forms  raised 
colonies  of  a  yellowish  colour,  with  umbilication  of  the  centre, 
and  there  is  no  liquefaction  of  the  medium.  On  agar  the 
growth  assumes  a  reddish  colour;  the  organism  flourishes  well 
in  various  vegetable  infusions  in  which  the  actinomyces  does 
not  grow.  On  all  the  media  growth  only  takes  place  in  aerobic 
conditions.  Experimental  inoculation  of  various  animals  has 
failed  to  reproduce  the  disease.  There  is  therefore  no  doubt 
that  the  streptothrix  madurae  and  the  actinomyces  are  distinct 
species. 

Black  Variety.  —  There  has  been  considerable  discussion 
regarding  the  relation  of  this  variety  of  the  disease  to  that  just 


FIG.  106.  —  Streptothrix  madurce,  show- 
ing branching  filaments.  From  a  culture 
on  agar.  Stained  with  carbol-thionin-blue. 
X  looo. 


MADURA   DISEASE. 


299 


described.  Kanthack  considered  that  the  parasite  was  the  same 
in  both,  and  occurred  in  the  black  variety  in  a  degenerated  form. 
Boyce  and  Surveyor,  on  the  other  hand,  pointed  out  differences, 
and  especially  that  in  the  black  variety  the  parasite  is  more 
highly  organised  and  the  branching  filaments  are  thicker  ;  hence 
they  believed  that  it  belonged  to  the  hyphomycetes.  The  obser- 
vations of  J.  H.  Wright,  who  obtained  pure  cultures  of  a  hypho- 
mycete,  are  confirmatory  of  this  view.  The  pigment  may  be 
dissolved  by  soaking  the  granules  for  a  few  minutes  in  hypo- 
chlorite  of  sodium  solution,  and  the  granule  may  then  be  crushed 
out  beneath  a  cover-glass  and  examined  microscopically.  The 
black  granules  are  composed  of  a  somewhat  homogeneous 
ground-substance  impregnated  with  pigment,  and  in  this  there 
is  a  mycelium  of  thick  filaments  or  hyphae,  many  of  the  seg- 
ments of  which  are  swollen ;  at  the  periphery  the  hyphse  form 
.a  zone  with  radiate  arrangement.  In  many  of  the  older  gran- 
ules the  parasite  is  largely  degenerated  and  presents  an  amor- 
phous appearance.  Wright  planted  over  sixty  of  the  black 
granules  in  various  culture  media,  and  obtained  cultures  of  a 
hyphomycete  from  about  a  third  of  these.  The  organism  grows 
well  on  agar,  bouillon,  potato,  etc. ;  on  agar  it  forms  a  felted 
mass  of  greyish  colour,  and  in  old  cultures  black  granules  appear 
amongst  the  mycelium.  Microscopically  the  parasite  appears 
as  a  mycelium  of  thick  branching  filaments  with  delicate  trans- 
verse septa ;  in  the  older  threads  the  segments  become  swollen, 
so  that  strings  of  oval-shaped  bodies  result.  No  signs  of  spore 
formation  were  noted.  Inoculation  of  animals  with  cultures 
gave  negative  results,  as  did  also  direct  inoculation  with  the 
black  granules  from  the  tissues. 


CHAPTER   XIV. 
ANTHRAX.1 

OTHER     NAMES  :  —  SPLENIC     FEVER,     MALIGNANT     PUSTULE, 
WOOLSORTER'S  DISEASE.     GERMAN,  MILZBRAND  ;  FRENCH, 

CHARBON.2 

Introductory.  —  Anthrax  is  a  disease  occurring  epidemically 
among  the  herbivora,  especially  sheep  and  oxen,  in  which  ani- 
mals it  has  the  characters  of  a  rapidly  fatal  form  of  septicaemia, 
with  splenic  enlargement,  attended  by  an  extensive  multiplication 
of  characteristic  bacilli  throughout  the  blood.  The  disease  does 
not  occur  as  a  natural  affection  in  man,  but  may  be  communi- 
cated to  him  directly  or  indirectly  from  animals,  and  it  may 
then  appear  in  certainly  two  and  possibly  three  forms.  In  the 
first  there  is  infection  through  the  skin,  in  which  a  local  lesion,, 
the  "malignant  pustule,"  occurs.  In  the  second  form  infection 
takes  place  through  the  respiratory  tract.  Here  very  aggravated 
symptoms  centred  in  the  thorax,  with  rapidly  fatal  termination, 
follow.  Thirdly,  an  infection  may  probably  take  place  through 
the  intestinal  tract,  which  is  now  the  first  part  to  give  rise  to 
symptoms.  In  all  these  forms  of  the  affection  in  the  human 
subject,  the  bacilli  are  in  their  distribution  much  more  restricted 
to  the  local  lesions  than  is  the  case  in  the  ox,  their  growth  and 
spread  being  attended  by  inflammatory  oedema  and  often  by 
haemorrhages. 

Historical  Summary.  —  Historical  researches  leave  little  doubt  that  from 
the  earliest  times  anthrax  has  occurred  among  cattle.  For  a  long  time  its 
pathology  was  not  understood,  and  it  went  by  many  names.  During  the 

1  In  even  recent  works  on  surgery  the  term  "anthrax"  may  be  found  applied 
to  any  form  of  carbuncle.     Before  its  true  pathology  was  known  the  local  variety 
of  the  disease  which  occurs  in  man  and  which  is  now  called  "  malignant  pustule  "  was- 
known  as  "  malignant  carbuncle." 

2  This  must  be  distinguished  from  "  charbon  symptomatique,"  which  is  quite  a- 
different  disease. 


BACILLUS   ANTHRACIS.  301 

early  part  of  the  present  century  much  attention  was  paid  to  it,  and,  with  a 
view  to  finding  out  its  nature  and  means  of  spread,  various  conditions  attach- 
ing to  its  occurrence,  such  as  those  of  soil  and  weather,  were  exhaustively 
studied.  Pollender  in  1849  pointed  out  that  the  blood  of  anthrax  animals 
contained  numerous  rod-shaped  bodies  which  he  conjectured  had  some  causal 
connection  with  the  disease.  In  1863  Davaine  announced  that  they  were 
bacteria,  and  originated  the  name  bacillus  anthracis.  He  stated  that  unless 
blood  used  in  inoculation  experiments  on  animals  contained  them,  death  did 
not  ensue.  Though  this  conclusion  was  disputed,  still  by  the  work  of  Davaine 
and  others  the  causal  relationship  of  the  bacilli  to  the  disease  had  been  nearly 
established  when  the  work  of  Koch  appeared  in  1876.  This  constituted  that 
observer's  first  contribution  to  bacteriology,  and  did  much  to  clear  up  the 
whole  subject.  Koch  confirmed  Davaine's  view  that  the  bodies  were  bacteria. 
He  observed  in  the  blood  of  anthrax  animals  the  appearance  of  division,  and 
from  this  deduced  that  multiplication  took  place  in  the  tissues.  He  observed 
them  under  the  microscope  dividing  outside  the  body,  and  noticed  spore 
formation  taking  place.  He  also  isolated  the  bacilli  in  pure  culture  outside 
the  body,  and  by  inoculating  animals  with  them,  produced  the  disease 
artificially.  In  his  earlier  experiments  he  failed  to  produce  death  by  feeding 
susceptible  animals  both  with  bacilli  and  spores,  and  as  the  intestinal  tract 
was,  in  his  view,  the  natural  path  of  infection,  he  considered  as  incomplete 
the  proof  of  this  method  of  the  spontaneous  occurrence  of  anthrax  in  herds 
of  animals.  Koch's  observations  were,  shortly  afterwards,  confirmed  in  the 
main  by  Pasteur,  though  controversy  arose  between  them  on  certain  minor 
points.  Moreover,  further  research  showed  that  the  disease  could  be  pro- 
duced in  animals  by  feeding  them  with  spores,  and  thus  the  way  in  which  the 
disease  might  spread  naturally  was  explained. 

The  Bacillus  Anthracis.  —  Anthrax  as  a  disease  in  man  is  of 
comparative  rarity.  Not  only,  however,  is  the  bacillus  anthracis 
easy  of  growth  and  recognition,  but  in  its  growth  it  illustrates 
many  of  the  general  morphological  characters  of  the  whole  group 
of  bacilli,  and  is  therefore  of  the  greatest  use  to  the  student. 
Further,  its  behaviour  when  inoculated  in  animals  illustrates 
many  of  the  points  raised  in  connection  with  such  difficult 
questions  as  the  general  pathogenic  effects  of  bacteria,  immunity, 
etc.  Hence  an  enormous  amount  of  work  has  been  done  in 
investigating  it  in  all  its  aspects. 

If  a  drop  of  blood  is  taken  immediately  after  death  from  an 
auricular  vein  of  a  cow,  for  example,  which  has  died  from 
anthrax,  and  examined  microscopically,  it  will  be  found  to 
contain  a  great  number  of  large  non-motile  bacilli.  On  making 
a  cover-glass  preparation  from  the  same  source,  and  staining  with 
watery  methylene-blue,  the  characters  of  the  bacilli  can  be  better 
made  out.  They  are  about  1.2  JJL  thick  or  a  little  thicker,. and 


302 


ANTHRAX. 


FlG.  107.  —  Surface  colony  of  the  anthrax 
bacillus  on  an  agar  plate,  showing  the  charac- 
teristic appearances.  X  30. 


6  to  8  //.  long,  though  both  shorter  and  longer  forms  also  occur. 
The  ends  are  sharply  cut  across,  or  may  be  slightly  dimpled  so 
as  to  resemble  somewhat  the  proximal  end  of  a  phalanx.  Their 

protoplasm  is  very  finely  granu- 
lar, and  sometimes  appears  sur- 
rounded by  a  thin  unstained 
capsule.  When  several  bacilli 
lie  end  to  end  in  a  thread,  the 
capsule  seems  common  to  the 
whole  thread  (Fig.  1 1 1 ).  They 
stain  well  with  all  the  basic 
aniline  dyes  and  are  not  de- 
colorised by  Grain's  method. 

Plate-cultures.  —  From  a 
source  such  as  that  indicated, 
it  is  easy  to  isolate  the  bacilli 
by  making  gelatin  or  agar 
plates.  If,  after  twelve  hours' 
incubation  at  37° C.,  the  latter  be  examined  under  a  low  objec- 
tive, colonies  will  be  observed.  They  are  to  be  recognised  by 
beautiful  wavy  wreaths,  like  locks  of  hair,  radiating  from  the 
centre  and  apparently  terminating  in  a  point,  which,  however, 
on  examination  with  a  higher 
power  is  observed  to  be  a  fila- 
ment which  turns  upon  itself 
(Fig.  107).  The  whole  colony 
is,  in  fact,  probably  one  long 
thread.  Such  colonies  are  very 
suitable  for  making  impression 
preparations  (vide  p.  114)  which 
preserve  permanently  the  ap- 
pearances described.  On  ex- 
amining such  with  a  high  power, 
the  wreaths  are  seen  to  be  made 
up  of  bundles  of  long  filaments 
lying  parallel  with  one  another, 
each  filament  consisting  of  a 
chain  of  bacilli  lying  end  to  end,  and  similar  to  those  observed 
in  the  blood  (Fig.  108). 

On  gelatin  plates,  after  from  twenty-four  to  thirty-six  hours 


FIG.  108.  —  Anthrax  bacilli,  arranged 
in  chains  from  a  twenty-four  hours'  culture 
on  agar  at  37°  C.  Stained  with  fuchsin. 
Xiooo. 


APPEARANCES  OF  CULTURES.  303 

at  20°  C.,  the  same  appearances  manifest  themselves,  and  later 
they  are  accompanied  by  liquefaction  of  the  gelatin.  In  gelatin 
plates,  however,  instead  of  the  characteristically  wreathed  ap- 
pearance at  the  margin,  the  colonies  sometimes  give  off  radiating 
spikelets  irregularly  nodulated,  which  produce  a  star-like  form. 
These  spikelets  are  composed  of  spirally  twisted  threads. 

From  such  plates  the  bacilli  can  be  easily  isolated,  and  the 
appearances  of  pure  cultures  on  various  media  studied. 

Appearances   of  Cultures.  —  In    botiillon,    after    twenty-four 
hours'  incubation  at  37°  C.,  there  is  usually  the  appearance  of 
irregularly  spiral  threads  or  flocculi  suspended  in  the  liquid  or 
lying  at  the  bottom  of  the  tube.    These,  on  being 
examined,  are  seen  to  be  made  up  of  bundles 
of  parallel  chains  of  bacilli.     Later,  growth  is 
more  abundant,  and  forms  a  flocculent  mass  at 
the  bottom  of  the  fluid. 

In  gelatin  stab-cultures,  the  characteristic 
appearance  can  be  best  observed  when  a  low 
proportion,  say  ;|  per  cent,  of  gelatin  is  present, 
and  when  the  tube  is  directly  inoculated  from 
anthrax  blood.  In  about  two  days  there  radi- 
ate out  into  the  medium  from  the  needle  track' 
numberless  very  fine  spikelets  which  enable  the 
cultures  to  be  easily  recognised.  These  spikelets 
are  longest  at  the  upper  part  of  the  needle  track 
(Fig.  109).  Not  much  spreading  takes  place  on 
the  surface  of  the  gelatin,  but  here  liquefaction 
commences,  and  gradually  spreads  down  the 
stab  and  out  into  the  medium,  till  the  whole  of 
the  gelatin  may  be  liquefied.  Gelatin  sloped  cul-  cul^eo'?heanfhrt 
tures  exhibit  a  thick  felted  growth,  the  edges  bacillus  in  peptone- 
of  which  show  the  wreathed  appearance  seen  in  growth'  itshows^he 
plate-cultures.  Liquefaction  here  soon  ploughs  "spiking"  and  also, 

.     .       ^  .  r  at  the  surface,  com- 

a  trough  in  the  surface  of  the  medium.  Some-  mencing  Hquefac- 
times  "  spiking  "  does  not  take  place  in  gelatin  tion-  Natural  size, 
stab-cultures,  only  little  round  particles  of  growth  occurring  down 
the  needle  track,  followed  by  liquefaction.  As  has  been  shown 
by  Richard  Muir,  this  property  of  spiking  can  be  restored  by 
growing  the  bacillus  for  twenty-four  hours  on  blood  agar  at 
37°C.  Agar  sloped  cultures  have  the  appearance  of  similar 


304  ANTHRAX. 

cultures  in  gelatin,  though,  of  course,  no  liquefaction  takes  place. 
Blood  serum  sloped  cultures  present  the  same  appearances  as 
those  on  agar.  The  margin  of  the  surface  growth  on  any  of  the 
solid  media  shows  the  characteristic  wreathing  seen  in  plate 
colonies.  On  potatoes  there  occurs  a  thick  felted  white  mass 
of  bacilli  showing  no  special  characters.  Such  a  growth,  how- 
ever, is  useful  for  studying  sporulation.  Litmus  milk  is  feebly 
acidified,  loosely  coagulated,  and  slowly  peptonised. 

The  anthrax  bacillus  will  thus  grow  readily  on  any  of  the 
ordinary  media.  It  can  usually  be  sufficiently  recognised  by  its 
microscopic  appearance,  by  its  growth  on  agar  or  gelatin  plates, 
and  by  its  growth  in  gelatin  stab-cultures.  The  growth  on 
plates  is  specially  characteristic,  and  is  simulated  by  no  other 
pathogenic  organism.  Among  the  non-pathogenic  bacteria  the 
only  organism  which  has  similar  colonies  is  the  bacillus  figurans, 
and  the  resemblance  is  only  a  distant  one.  One  variety  of 
B.  subtilis  has  been  observed  to  bear  a  striking  general  resem- 
blance to  B.  anthracis,  however. 

The  Biology  of  Bacillus  Anthracis.  —  Koch  found  that  the 
bacillus  anthracis  grows  best  at  a  temperature  of  35°  C.  Growth, 
i.e.  multiplication,  does  not  take  place  below  12°  C.  or  above 
45°  C.  In  the  spore-free  condition  the  bacilli  have  comparatively 
low  powers  of  resistance.  They  do  not  stand  long  exposure  to 
60°  C.,  and  if  kept  at  ordinary  temperature  in  the  dry  condition 
they  are  usually  found  to  be  dead  after  a  few  days.  The  action 
of  the  gastric  juice  is  rapidly  fatal  to  them,  and  they  are  accord- 
ingly destroyed  in  the  stomachs  of  healthy  animals.  They  are 
also  soon  killed  in  the  process  of  putrefaction.  They  can,  how- 
ever, be  cooled  below  the  freezing-point  without  dying.  The 
bacillus  can  grow  without  oxygen,  but  some  of  its  vital  functions 
are  best  carried  on  in  the  presence  of  this  gas.  Thus  in  anthrax 
cultures  the  liquefaction  of  gelatin  always  commences  at  the 
surface  and  spreads  downwards.  Growth  is  more  rapid  in  the 
presence  of  oxygen,  and  spore  formation  does  not  occur  in 
its  absence.  The  organism  may  be  classed  as  a  facultative 
anaerobe. 

Sporulation.  —  Under  certain  circumstances  sporulation  oc- 
curs in  anthrax  bacilli.  The  morphological  appearances  are  of 
the  ordinary  kind.  A  little  highly  refractile  speck  appears  in 
the  protoplasm  about  the  centre  of  the  bacillus  ;  this  gradually 


SPORULATION    OF   B.    ANTHRACIS. 


305 


increases  in  size  until  it  forms  an  oval  body,  about  the  same 
thickness  as  the  bacillus,  lying  in  the  bacillary  protoplasm  (Fig. 
no).  The  latter  gradually  loses  its  staining  capacities  and 
finally  disappears.  The  spore  thus  lies  free  as  an  oval  highly 
refractile  body  which  does  not  stain  by  ordinary  methods,  but 
which  can  be  easily  stained  by  the  special  methods  described 
for  such  a  purpose  (p.  106).  When  the  spore  is  again  about  to 
assume  the  bacillary  form  the  capsule  is  apparently  absorbed, 
and  the  protoplasm  within  grows  out,  taking  on  the  ordinary 
rod-shaped  form. 

According  to  most  observers  sporulation  never  occurs  within 
the  body  of  an  animal  suffering  from  anthrax.  Koch  attributes 
this,  probably  rightly,  to  the 
absence  of  free  oxygen.  The 
latter  gas  he  found  necessary  to 
the  occurrence  of  spores  in  cul- 
tures outside  the  body.  Many, 
however,  are  inclined  to  assign 
as  the  cause  of  sporulation  the 
absence  of  the  optimum  pabu- 
lum, which  in  the  case  of  an- 
thrax is  afforded  by  the  animal 
tissues.  Besides  these  condi- 
tions there  is  another  factor 
necessary  to  sporulation,  viz., 
a  suitable  temperature.  The 
optimum  temperature  for  spore 
production  is  32°  C.  Koch 
found  that  spore  formation  did  not  occur  below  i8°-C.  Above 
42°  C.  not  only  does  sporulation  cease,  but  Pasteur  found  that 
if  bacilli  were  kept  at  this  temperature  for  eight  days  they  did 
not  regain  the  capacity  when  again  grown  at  a  lower  tempera- 
ture. In  order  to  make  them  again  capable  of  sporing  it  is 
necessary  to  adopt  special  measures,  such  as  passage  through 
the  bodies  of  a  series  of  susceptible  animals. 

Anthrax  spores  have  extremely  high  powers  of  resistance. 
In  a  dry  condition  they  will  remain  viable  for  a  year  or  more. 
Koch  found  they  resisted  boiling  for  five  minutes ;  and  dry  heat 
at  140°  C.  must  be  applied  for  several  hours  to  kill  them  with 
certainty.  Unlike  the  bacilli,  they  can  resist  the  action  of  the 


FIG.  no.  —  Anthrax  bacilli  containing 
spores  (the  darkly  coloured  bodies)  ;  from  a 
three  days'  culture  on  agar  at  37°  C. 

Stained  with  carbol-fuchsin  and  methy- 
lene-blue.  X  1000. 


306  ANTHRAX. 

gastric  juice  for  a  long  period  of  time.  They  are  often  used 
as  test  objects  by  which  the  action  of  germicides  is  judged.  For 
this  purpose  an  emulsion  is  made  by  scraping  off  a  surface 
culture  and  rubbing  it  up  in  a  little  sterile  water.  Into  this 
sterile  silk  threads  are  dipped,  which,  after  being  dried  over 
strong  sulphuric  acid  in  a  desiccator,  can  be  kept  for  long 
periods  of  time  in  an  unchanged  condition.  For  use  they  are 
placed  in  the  germicidal  solution  for  the  desired  time,  then 
washed  with  water  to  remove  the  last  traces  of  the  reagent  and 
laid  on  the  surface  of  agar  or  placed  in  bouillon,  in  order  that  if 
death  has  not  occurred  growth  may  be  observed. 

Anthrax  in  Animals.  —  Anthrax  occurs  from  time  to  time 
epidemically  in  sheep,  cattle,  and,  more  rarely,  in  horses  and 
deer.  These  epidemics  are  found  in  various  parts  of  the  world, 
although  they  are  naturally  most  far-reaching  where  legal  pre- 
cautions to  prevent  the  spread  of  infection  are  non-existent. 
All  the  countries  of  Europe  are  from  time  to  time  visited  by  the 
disease,  but  in  some  it  is  much  more  common  than  in  others. 
In  Britain  the  death-rate  is  small,  but  in  France  the  annual 
mortality  among  sheep  was  probably  10  per  cent  of  the  total 
number  in  the  country,  and  among  cattle  5  per  cent.  These 
figures,  however,  have  been  largely  modified  by  the  system  of  pre- 
ventive treatment  which  will  be  presently  described.  In  sheep 
and  cattle  the  disease  is  specially  virulent.  An  animal  may 
suddenly  drop  down,  with  symptoms  of  collapse,  quickening  of 
pulse  and  respiration,  and  dyspnoea,  and  death  may  occur  in 
a  few  minutes.  In  less  acute  cases  the  animal  is  apparently 
out  of  sorts,  and  does  not  feed ;  its  pulse  and  respiration  are 
quickened  ;  rigours  occur,  succeeded  by  high  temperature ;  there 
is  a  sanguineous  discharge  from  the  bowels,  and  bloody  mucus 
may  be  observed  about  the  mouth  and  nose.  There  may  be 
convulsive  movements,  there  is  progressive  weakness,  with  cyano- 
sis, death  occurring  in  from  twelve  to  forty-eight  hours.  In  the 
more  prolonged  cases  widespread  oedema  and  extensive  enlarge- 
ment of  lymphatic  glands  are  marked  features  ;  and  in  the  glands, 
especially  about  the  neck,  actual  necrosis  with  ulceration  may 
occur,  constituting  the  so-called  anthrax  carbuncles.  Such 
subacute  conditions  are  especially  found  among  horses,  which 
are  by  nature  not  so  susceptible  to  the  disease  as  cattle  and 
sheep. 


ANTHRAX   IN   ANIMALS. 


307 


On  post-mortem  examination  of  an  ox  dead  of  anthrax,  the 
most  noticeable  feature — one  which  has  given  the  name  "splenic 
fever"  to  the  disease — is  the  enlargement  of  the  spleen,  which 
may  be  two  or  three  times  its  natural  size.  It  is  of  dark-red  colour, 
and  on  section  the  pulp  is  very  soft  and  friable,  sometimes  almost 
diffluent.  A  cover-glass  preparation  may  be  made  from  the 
spleen  and  stained  with  watery  methylene-blue.  On  examina- 
tion it  will  be  found  to  contain  enormous  numbers  of  bacilli 
mixed  with  red  corpuscles  and  leucocytes,  chiefly  lymphocytes 
and  the  large  mononucleated  variety  (Fig.  in).  Pieces  of  the 
organ  may  be 
hardened  in  ab- 
solute alcohol, 
and  sections 
cut  in  paraf- 
fin. These  are 
best  stained  by 
Gram's  meth^ 
o  d.  Micro- 
scopic  exami- 
nation of  such 
shows  that  the 
structure  of  the 
pulp  is  con- 
siderably dis- 
integrated, 
whilst  the  ba- 
cilli swarm 
throughout  the 
organ,  lying 
irregularly 
amongst  the 

cellular  elements.  The  liver  is  enlarged  and  congested,  and  may 
be  in  a  state  of  acute  cloudy  swelling.  The  bacilli  are  present 
in  the  capillaries  throughout  the  organ,  but  are  not  so  numerous 
as  in  the  spleen.  The  kidney  is  in  a  similar  condition,  and  here 
the  bacilli  are  chiefly  found  in  the  capillaries  of  the  glomeruli, 
which  often  appear  as  if  injected  with  them.  The  lungs  are 
congested  and  may  show  catarrh,  whilst  bacilli  are  present  in 
large  numbers  throughout  the  capillaries,  and  may  also  be  found 


-.»*. 


FIG.  in.  —  Scraping  from  spleen  of  guinea-pig  dead  of  anthrax, 
showing  the  bacilli  mixed  with  leucocytes,  etc.  (Same  appearance 
as  in  the  ox.) 

"  Corrosive  film  "  stained  with  carbol-thionin-blue.     X  1000. 


308  ANTHRAX. 

in  the  air  cells,  probably  as  the  result  of  rupture  of  the  capil- 
laries. The  blood  throughout  the  body  is  usually  fluid  and  of 
dark  colour. 

The  lymphatic  system  generally  is  much  affected.  The 
glands,  especially  the  mediastinal,  mesenteric,  and  cervical 
glands,  are  enlarged  and  surrounded  by  oedematous  tissue,  the 
lymphatic  vessels  are  swollen,  and  both  glands  and  vessels  may 
contain  numberless  bacilli.  The  heart  may  be  in  a  state  of 
cloudy  swelling,  and  the  blood  in  its  cavities  contains  bacilli, 
though  in  smaller  numbers  than  that  in  the  capillaries.  The 
intestines  are  enormously  congested,  the  epithelium  more  or  less 
desquamated,  and  the  lumen  filled  with  a  bloody  fluid.  From 
all  the  organs  the  bacilli  can  be  easily  isolated  by  stroke-cultures 
on  agar. 

It  is  important  to  note  the  existence  of  great  differences  in 
susceptibility  to  anthrax  in  different  species  of  animals.  Thus 
the  ox,  sheep  (except  those  of  Algeria,  which  only  succumb  to 
enormous  doses  of  the  bacilli),  guinea-pig,  and  mouse  are  all 
very  susceptible,  the  rabbit  slightly  less  so.  The  last  three  are 
of  course  most  used  for  experimental  inoculation.  We  have  no 
data  to  determine  whether  the  disease  occurs  among  these  in  the 
wild  state.  Less  susceptible  than  this  group  are  the  horse,  deer, 
goat,  in  which  the  disease  occurs  from  time  to  time  in  nature. 
Anthrax  also  occurs  epidemically  in  the  pig,  often  from  the 
ingestion  of  the  organs  of  other  animals  dead  of  the  disease. 
It  is,  however,  doubtful  if  all  cases  of  disease  in  the  pig  described 
on  clinical  grounds  as  anthrax  are  really  such,  and  a  careful 
bacteriological  examination  is  always  advisable.  The  human 
subject  may  be  said  to  occupy  a  medium  position  between  the 
highly  susceptible  and  the  relatively  immune  animals.  The  white 
rat  is  highly  immune  to  the  disease,  while  the  brown  rat  is  sus- 
ceptible. Adult  carnivora  are  also  very  immune,  and  the  birds 
and  amphibia  are  in  the  same  position. 

With  these  differences  in  susceptibility  there  are  also  great 
variations  in  the  pathological  effects  produced  in  the  natural  or 
artificial  disease.  This  is  especially  the  case  when  we  consider 
the  distribution  of  the  bacilli  in  the  bodies  of  the  less  susceptible 
animals.  Instead  of  the  widespread  occurrence  described  above, 
they  may  be  confined  to  the  point  where  they  first  gained  access 
to  the  body  and  the  lymphatic  system  in  relation  to  it,  or  may 


EXPERIMENTAL   INOCULATION. 


309 


be  only  very  sparsely  scattered  in  organs  such  as  the  spleen 
(which  is  often  not  enlarged),  the  lungs,  or  kidneys.  Neverthe- 
less the  cellular  structure  of  the  organs  even  in  such  a  case  may 
show  changes,  a  fact  which  is  important  when  we  consider  the 
essential  pathology  of  the  disease. 

Experimental  Inoculation.  —  Of  the  animals  commonly  used 
in  laboratory  work,  white  mice  and  guinea-pigs  are  the  most: 
susceptible  to  anthrax,  and  are  generally  used  for  test  inocula- 
tions. If  a  small  quantity  of  anthrax  bacilli  be  injected  into  the 
subcutaneous  tissue  of  a  guinea-pig  a  fatal  result  follows,  usually 
within  two  days.  Post  mortem  around  the  site  of  inoculation  the 
tissues,  owing  to 

intense     inflam-  ^  ' 

matory  oedema, 
are  swollen  and 
gelatinous  in  ap- 
pearance, small 
haemorrhages 
are  often  pres- 
ent, and  on  mi- 
croscopic exami- 
nation numerous 
bacilli  are  seen. 
The  internal 
organs  show 
congestion  and 
cloudy  swelling, 
with  sometimes  "**  % 

small      haemor- 

,  ji.  FIG.  112.  —  Portion  of  kidney  of  a  guinea-pig  dead  of  anthrax, 

rnageS,  and  their  showing  the  bacilli  in  the  capillaries,  especially  of  the  glomeru- 

CapillarieS       COn-  lus'     Paraffin  section  ;  stained  by  Gram's  method  and  Bismarck- 

.  brown.       XSOQ. 

tain      enormous 

numbers  of  bacilli,  as  has  already  been  described  in  the  case  of 
the  ox  (Fig.  112);  the  spleen  also  shows  a  corresponding  condi- 
tion. Highly  susceptible  animals  may  be  infected  by  being  made 
to  inhale  the  bacilli  or  their  spores,  and  also  by  being  fed  with 
spores,  a  general  infection  rapidly  occurring  by  both  methods. 

Anthrax  in  the  Human  Subject.  —  As  we  have  noted,  man 
occupies  a  middle  position  in  the  scale  of  susceptibility  to 
anthrax.  It  is  always  communicated  to  him  from  animals, 


3IO  ANTHRAX. 

and  usually  is  seen  among  those  whose  trade  leads  them  to 
handle  the  carcases  or  skins  of  animals  which  have  died  of  the 
disease.  It  occurs  in  two  principal  forms,  the  main  difference 
between  which  is  the  site  of  entrance  of  the  organism  into  the 
body.  In  one,  the  path  of  entrance  is  through  cuts  or  abrasions 
in  the  skin,  or  through  the  hair  follicles.  A  local  condition 
called  a  "  malignant  pustule"  develops,  which  may  lead  to  a 
general  infection.  This  variety  occurs  chiefly  among  butchers 
and  those  who  work  among  hides  (foreign  ones  especially).  In 
Britain  the  workers  of  the  latter  class  chiefly  liable  are  the  hide- 
porters  and  hide-workers  in  South-eastern  London.  In  the 
other  variety  of  the  disease  the  site  of  infection  is  the  trachea 
and  bronchi,  and  here  a  fatal  result  almost  always  follows.  The 
cause  is  the  inhalation  of  dust  or  threads  from  wool,  hair,  or 
bristles,  which  have  been  taken  from  animals  dead  of  the 
disease,  and  which  have  been  contaminated  with  blood  or  secre- 
tions containing  the  bacilli,  these  having  afterwards  formed 
spores.  This  variety  is  often  referred  to  as  "  woolsorter's  dis- 
ease," from  its  occurring  in  the  centres  of  the  woolstapling  trade 
(in  England,  chiefly  in  Yorkshire),  but  it  also  is  found  in  places 
where  there  are  hair  and  brush  factories. 

(i)  Malignant  Pustule.  — This  usually  occurs  on  the  exposed 
surfaces,  —  the  face,  hands,  forearms,  and  back,  the  last  being  a 
common  site  among  hide-porters.  One  to  three  days  after  inoc- 
ulation a  small  red  painful  pimple  appears,  soon  becoming  a 
vesicle,  which  may  contain  clear  or  blood-stained  fluid,  and  is 
rapidly  surrounded  by  an  area  of  intense  congestion.  Central 
necrosis  occurs  and  leads  to  the  malignant  pustule  proper, 
which  in  its  typical  form  appears  as  a  black  eschar  often  sur- 
rounded by  a  ring  of  vesicles,  these  in  turn  being  surrounded 
by  a  congested  area.  From  this  pustule  as  a  centre  subcutaneous 
oedema  spreads,  especially  in  the  direction  of  the  lymphatics ; 
the  neighbouring  glands  are  enlarged.  There  is  fever  with 
general  malaise.  On  microscopic  section  of  the  typical  pustule, 
the  central  eschar  is  noticed  to  be  composed  of  necrosed  tissue 
and  degenerating  blood  cells ;  the  vesicles  are  formed  by  the 
raising  of  the  stratum  corneum  from  the  rete  Malpighi.  Be- 
neath them  and  in  their  neighbourhood  the  cells  of  the  latter 
are  swollen  and  oedematous,  the  papillae  being  enlarged  and 
flattened  out  and  infiltrated  with  inflammatory  exudation,  which 


WOOLSORTER'S   DISEASE.  311 

also  extends  beneath  the  centre  of  the  pustule.  In  the  tissue 
next  the  eschar  necrosis  is  commencing.  The  subcutaneous 
tissue  is  also  oedematous,  and  often  infiltrated  with  leucocytes. 
The  bacilli  exist  in  the  periphery  of  the  eschar  and  in  the  neigh- 
bouring lymphatics,  and,  to  a  certain  extent,  in  the  vesicles.  It 
is  very  important  to  note  that  widespread  oedema  of  a  limb, 
enlargement  of  neighbouring  glands,  and  fever  may  occur  while 
the  bacilli  are  still  confined  to  the  immediate  neighbourhood 
of  the  pustule.  Sometimes  the  pathological  process  goes  no 
further,  the  eschar  becomes  a  scab,  the  inflammation  subsides, 
and  recovery  takes  places.  In  the  majority  of  cases,  however, 
if  the  pustule  be  not  excised,  the  oedema  spreads,  invasion  of 
the  blood  stream  may  occur,  and  the  patient  dies  with,  in  a 
modified  degree,  the  pathological  changes  detailed  with  regard 
to  the  acute  disease  in  cattle.  In  man  the  spleen  is  usually  not 
much  enlarged,  and  the  organs  generally  contain  few  bacilli. 
The  actual  cause  of  death  is  therefore  the  absorption  of  toxins. 
It  may  here  be  said  that  early  excision  of  an  anthrax  pustule, 
especially  when  it  is  situated  in  the  extremities,  is  followed,  in 
a  large  proportion  of  cases,  by  recovery. 

(2)  Woolsorter's  Disease.  —  The  pathology  of  this  affection 
was  worked  out  in  this  country  especially  by  Greenfield.  The 
local  lesion  is  usually  situated  in  the  lower  part  of  the  trachea 
or  in  the  large  bronchi,  and  is  in  the  form  of  swollen  patches  in 
the  mucous  membrane  often  with  haemorrhage  into  them.  The 
tissues  are  oedematous,  and  the  cellular  elements  are  separated, 
but  there  is  usually  little  or  no  necrosis.  There  is  enormous 
enlargement  of  the  mediastinal  and  bronchial  glands,  and 
haemorrhagic  infiltration  of  the  cellular  tissue  in  the  region. 
There  are  pleural  and  pericardial  effusions,  and  haemorrhagic 
spots  occur  beneath  the  serous  membranes.  The  lungs  show 
collapse  and  oedema.  There  may  be  cutaneous  oedema  over 
the  chest  and  neck,  with  enlargement  of  glands,  and  the  patient 
rapidly  dies  with  symptoms  of  pulmonary  embarrassment,  and 
with  a  varying  degree  of  pyrexia.  It  is  to  be  noted  that  in 
such  cases,  though  numerous  bacilli  are  present  in  the  bronchial 
lesions,  in  the  lymphatic  glands,  and  affected  tissues  in  the 
thorax,  comparatively  few  may  be  present  in  the  various  organs, 
such  as  the  kidney,  spleen,  etc.,  and  sometimes  it  may  be 
impossible  to  find  any. 


312  ANTHRAX. 

(3)  Intestinal  Infection.  —  It  is  probable  that  infection  occa- 
sionally takes  place  through  the  intestine  ;  but  this  condition  is 
rare.  In  such  cases  there  is  a  local  lesion  in  the  intestinal 
mucous  membrane,  of  similar  nature  to  that  in  the  bronchial 
form,  with  a  corresponding  affection  of  the  mesenteric  glands. 

The  Toxins  of  the  Bacillus  Anthracis.  —  Various  theories 
were  formerly  held  as  to  the  mode  in  which  the  anthrax  bacillus 
produces  its  effects.  One  of  the  earliest  was  the  mechanical, 
according  to  which  it  was  supposed  that  the  serious  results  were 
produced  by  extensive  blocking  of  the  capillaries  in  the  various 
organs  by  the  bacilli.  According  to  another,  it  was  supposed 
that  the  bacilli  used  up  the  oxygen  of  the  blood,  thus  leading  to 
starvation  of  the  tissues.  Though  such  modes  of  action  may 
occur  to  a  small  extent,  we  now  know  that  in  anthrax,  as  in 
other  diseases,  the  important  local  and  general  effects  are 
produced  by  specific  poisons  formed  by  the  bacilli.  We  have 
therefore  to  consider  the  nature  of  these  toxic  bodies. 

During  the  years  1889-90  several  papers  were  published 
dealing  with  the  toxins  of  the  bacillus  anthracis.  Hankin, 
investigating  the  means  of  conferring  immunity  against  the 
disease,  isolated  from  cultures  in  a  bouillon  made  from  Liebig's 
meat  juice  an  albumose  which  he  considered  to  be  the  toxin. 
His  reason  for  thinking  so  was  that,  while  the  injection  of  very 
small  doses  of  this  substance  (one  five-millionth  to  one  ten- 
millionth  of  the  weight  of  an  animal)  lengthened  the  incubation 
period  of  the  disease,  and  might  even  ward  off  a  fatal  attack,  the 
injection  of  larger  doses  hastened  the  death  of  the  animal. 
Very  full  researches  on  the  subject  were  carried  out  by  Sidney 
Martin.  This  observer  used  alkali-albumin  on  which  to  grow 
the  bacillus,  this  medium  approaching  most  closely  to  the 
environment  of  the  latter  when  growing  in  the  animal  body. 
From  cultures  in  this  medium,  concentrated  by  evaporation 
either  at  100°  C.  or  in  vacuo  at  35°  to  45°  C.,  there  were  iso- 
lated proto-albumose,  deutero-albumose,  and  traces  of  peptone. 
The  albumoses  differed  from  those  which  occur  in  ordinary 
digestion,  in  being  strongly  alkaline  in  their  reaction.  This 
alkalinity,  Martin  held,  was  due  to  traces  of  an  alkaloidal  body 
of  which  the  albumoses  were  the  precursors,  and  which  were 
formed  when  the  process  of  digestion  of  the  alkali-albumin  by 
the  bacillus  was  allowed  to  go  on  further.  By  the  albumoses 


THE   TOXINS   OF    BACILLUS   ANTHRACIS.  313 

and  the  alkaloid,  pathogenic  effects  were  produced  in  animals 
closely  similar  to  those  produced  by  the  bacilli  themselves. 
Martin  adduced  evidence  to  show  that,  of  the  symptoms  of  the 
disease,  the  fever  was  mostly  due  to  the  albumoses,  while  the 
oedema  and  congestion  were  mostly  due  to  the  alkaloid,  which 
acted  as  a  local  irritant.  He  showed  that  prolonged  boiling 
destroyed  the  activity  of  the  albumoses,  but  not  that  of  the 
alkaloid.  Further,  from  the  body  fluids  of  animals  dead  of 
anthrax  he  isolated  poisonous  bodies  similar  to  those  produced 
by  the  bacilli  growing  in  this  artificial  medium.  Hankin,  in  a 
later  research  with  Wesbrook,  arrived  at  the  conclusion  that 
the  bacillus  anthracis  produces  a  ferment  which,  diffusing  out 
into  the  culture  fluid,  elaborates  albumoses  from  the  proteids 
present  in  it.  The  bacilli  also  produce  albumoses  directly  with- 
out the  intervention  of  a  ferment.  The  albumoses  produced  in 
the  latter  way,  when  injected  in  small  doses,  cause  in  susceptible 
animals  immunity  against  subsequent  inoculation  with  virulent 
bacilli,  but  are  only  toxic  to  animals  not  very  susceptible  to  the 
disease.  Marmier,  after  cultivating  the  B.  anthracis  in  peptone 
solution  containing  certain  salts,  removed  all  the  albumoses  from 
the  resultant  liquid,  and  from  them,  either  by  dialysis  or  extrac- 
tion with  glycerin,  isolated  a  body  which  gave  no  reactions  of 
albuminoid  matter,  peptone,  propeptone,  or  alkaloid.  This  he 
considers  the  toxin.  It  killed  animals  susceptible  to  anthrax  by 
a  sort  of  cachexia,  and  in  suitably  small  doses  could  be  used  to 
immunise  them  against  subsequent  inoculation  with  virulent 
bacilli.  It  was  chiefly  retained  within  the  bacilli  when  these 
were  growing  in  the  most  favourable  conditions.  Unlike  the 
toxins  of  tetanus  and  diphtheria,  and  unlike  ferments,  it  was 
not  destroyed  by  heating  to  110°  C.  The  toxin  produced  by 
the  B.  anthracis  growing  in  a  fluid  medium  remains  intimately 
associated  with  the  bacterial  protoplasm,  as  such  cultures  when 
filtered  are  relatively  non-toxic. 

From  this  account  of  the  researches  into  the  toxins  of  the 
B.  anthracis,  it  will  be  seen  that  our  knowledge  is  far  from  com- 
plete. It  is  difficult  to  say  what  interpretation  is  to  be  put 
on  the  results  of  Hankin  and  Wesbrook.  The  researches  of 
Marmier  rather  indicate  that,  as  is  the  case  with  the  toxins  of 
other  bacilli,  the  toxin  of  anthrax  may  belong  to  a  group  of  non- 
proteid  bodies  of  whose  chemical  nature  we  are  in  complete 


314  ANTHRAX. 

ignorance.  Be  this  as  it  may,  the  results  detailed  open  up  a 
way  for  our  arriving  at  an  idea  of  the  true  pathology  of  the 
disease.  The  bacilli  in  all  parts  of  the  body,  whether  directly 
or  intermediately  by  ferments,  produce  bodies  toxic  to  tissue 
cells.  Further,  bacilli  confined  locally  produce  by  this  means 
effects  on  distant  tissues.  This  explains  how  in  certain  cases, 
while  the  bacilli  are  still  locally  confined,  there  may  occur 
oedema  spreading  from  the  pustule,  and  pyrexia. 

The  Spread  of  the  Disease  in  Nature.  —  We  have  seen  that 
the  B.  anthracis  rarely,  if  ever,  forms  spores  in  the  body,  and  if 
the  bacilli  could  be  confined  to  the  blood  and  tissues  of  carcases 
of  animals  dying  of  the  disease,  it  is  certain  that  anthrax  in  an 
epidemic  form  would  rarely  occur.  For  it  has  been  shown  by 
many  observers  that  in  the  course  of  the  putrefaction  of  such  a 
carcase  the  anthrax  bacilli  rapidly  die  out,  and  that  after  ten 
days  or  a  fortnight  very  few  remain.  But  it  must  be  remem- 
bered that  while  still  alive,  an  animal  is  shedding  into  the  air  by 
the  bloody  excretions  from  the  mouth,  nose,  and  bowel,  myriads 
of  bacilli  which  may  rapidly  spore,  and  thus  arrive  at  a  very  re- 
sistant stage.  These  lie  on  the  surface  of  the  ground  and  are 
washed  off  by  surface  water.  At  certain  seasons  of  the  year 
the  temperature  is,  however,  sufficiently  high  to  permit  of  their 
germination,  and  also  of  their  multiplication,  as  they  can  un- 
doubtedly grow  on  the  organic  matter  which  occurs  in  nature. 
They  can  again  form  spores.  It  is  in  the  condition  of  spores 
that  they  are  dangerous  to  susceptible  animals.  In  the  bacillary 
stage,  if  swallowed,  they  will  be  killed  by  the  acid  gastric  con- 
tents; but  as  spores,  they  can  pass  uninjured  through  the 
stomach,  and,  gaining  an  entrance  into  the  intestine,  infect  its 
wall,  and  ultimately  reach,  and  multiply  in,  the  blood.  It  is 
known  that  in  the  great  majority  of  cases  of  the  disease  in 
sheep  and  oxen,  infection  takes  place  thus  from  the  intestine. 
It  was  thought  by  Pasteur  that  worms  were  active  agents  in  the 
natural  spread  of  the  disease  by  bringing  to  the  surface  anthrax 
spores.  Koch  made  direct  experiments  on  this  point,  and  could 
get  no  evidence  that  this  was  the  case.  He  thinks  it  much 
more  probable  that  the  recrudescence  of  epidemics  in  fields 
where  anthrax  carcases  have  been  buried  is  due  to  persistence 
of  spores  on  the  surface  which  has  been  infected  by  the  cattle 
when  alive. 


IMMUNISATION   AGAINST   ANTHRAX.  315 

The  Disposal  of  the  Carcases  of  Animals  dead  of  Anthrax.  —  It  is  extremely 
important  that  anthrax  carcases  should  be  disposed  of  in  such  a  way  as  to  pre- 
vent their  becoming  future  sources  of  infection.  If  anthrax  be  suspected  as 
the  cause  of  death  no  post-mortem  examination  should  be  made,  but  only  a 
small  quantify  of  blood  be  removed  from  an  auricular  vein  for  bacteriological 
investigation.  If  such  a  carcase  be  now  buried  in  a  deep  pit  surrounded  by 
quicklime,  little  danger  of  infection  will  be  run.  The  bacilli  being  confined 
within  the  body  will  not  spore,  and  will  die  during  the  process  of  putrefaction. 
The  danger  of  sporulation  taking  place  is,  of  course,  much  greater  when  an 
animal  has  died  of  an  unknown  disease  which  on  post-mortem  examination 
has  proved  to  be  anthrax,  but  similar  measures  for  burial  must  be  here 
adopted.  In  some  countries  anthrax  carcases  are  burned,  and  this,  if  practi- 
cable, is  of  course  the  best  means  of  treating  them.  The  chief  source  of 
danger  to  cattle  subsequently,  however,  proceeds  from  the  infection  of  fields, 
yards,  and  byres  with  the  offal,  and  the  discharge  from  the  mouths,  of  anthrax 
animals.  All  material  that  can  be  recognised  as  such  should  be  burned  along 
with  the  straw  in  which  the  animals  have  lain.  The  stalls  or  buildings  in 
which  the  anthrax  cases  have  been  must  be  limewashed.  Needless  to  say,  the 
greatest  care  must  be  taken  in  the  case  of  men  who  handle  the  animal  or  its 
carcase  that  they  have  no  wounds  on  their  persons,  and  that  they  thoroughly 
disinfect  themselves  by  washing  their  hands,  etc.,  in  I  to  1000  solution  of  cor- 
rosive sublimate,  and  that  all  clothes  soiled  with  blood,  etc.,  from  anthrax 
animals  be  thoroughly  boiled  or  steamed  for  half  an  hour  before  being  washed. 

The  Immunising  of  Animals  against  Anthrax.  —  Having  ascer- 
tained that  there  was  ground  for  believing  that  in  cattle  one 
attack  of  anthrax  protected  against  a  second,  Pasteur  (in  the 
years  1880-82)  elaborated  a  method  by  which  a  mild  form  of 
the  disease  could  be  given  to  animals,  which  rendered  harmless 
a  subsequent  inoculation  with  virulent  bacilli.  He  found  that 
the  continued  growth  of  anthrax  bacilli  at  42°  to  43°  C.  caused 
them  to  lose  their  capacity  of  producing  spores,  and  also  gradu- 
ally to  lose  their  virulence,  so  that  after  twenty-four  days  they 
could  no  longer  kill  either  guinea-pigs,  rabbits,  or  sheep.  Such 
cultures  constituted  his  premier  vaccin,  and  protected  against  the 
subsequent  inoculation  with  bacilli  which  had  been  grown  for 
twelve  days  at  the  same  temperature,  and  the  attenuation  of 
which  had  therefore  not  been  carried  so  far.  The  latter  consti- 
tuted the  deuxteme  vaccin.  It  was  further  found  that  sheep 
thus  twice  vaccinated  now  resisted  inoculation  with  a  culture 
which  usually  would  be  fatal.  The  method  was  to  inoculate  a 
sheep  on  the  inner  side  of  the  thigh  by  the  subcutaneous  injec- 
tion, from  a  hypodermic  syringe,  of  about  five  drops  of  the 
premier  vaccin ;  twelve  days  later  to  again  inoculate  with  the 


316  ANTHRAX. 

deuxttme  vaccin ;  fourteen  days  later  an  ordinary  virulent  cul- 
ture was  injected  without  any  ill  result.  This  method  was 
applicable  also  to  cattle  and  horses,  about  double  the  dose  of  each 
vaccine  being  here  necessary.  Extended  experiments  in  France 
generally  confirmed  earlier  results,  and  the  method  was,  before 
long,  used  to  mitigate  the  disease,  which  in  many  departments 
was  endemic  and  a  very  great  scourge.  Since  that  time  the 
method  has  been  regularly  in  use.  It  is  difficult  to  arrive  at  a 
certain  conclusion  as  to  its  ments.  Undoubtedly  a  certain  num- 
ber of  animals  die  of  anthrax  either  after  the  first  or  second  vac- 
cination, or  during  the  following  vaccination.  At  the  end  of  a 
year  the  immunity  is  lost  in  about  40  per  cent  of  the  animals 
vaccinated ;  and  thus  to  be  permanently  efficacious  the  process 
would  have  to  be  repeated  every  year.  Further,  the  immunity 
is  much  higher  in  degree  if,  after  the  first  and  second  vaccina- 
tions, an  inoculation  with  virulent  anthrax  is  performed.  Every- 
thing being  taken  into  account,  however,  there  is  no  doubt  that 
the  mortality  from  natural  anthrax  is  much  diminished  by  this 
system. 

Statistics  are  available  for  the  twelve  years  1882-93.  During  that  time 
3,296,815  sheep  were  vaccinated,  with  a  mortality,  either  after  the  first  or 
second  vaccination,  or  during  the  subsequent  twelve  months,  of  .94  per  cent, 
as  contrasted  with  the  ordinary  mortality  in  all  the  flocks  of  the  districts  of  10 
per  cent.  During  the  same  time  438,824  cattle  were  vaccinated,  with  a  mor- 
tality of  .34  per  cent,  as  contrasted  with  a  probable  mortality  of  5  per  cent 
if  they  had  been  unprotected. 

Other  means  of  immunising  animals  against  anthrax  have 
been  elaborated,  but  these  have  a  more  strictly  scientific  interest. 
In  dealing  with  the  toxins  of  anthrax  we  have  already  referred 
to  the  work  of  Hankin  and  Wesbrook  on  this  point.  We  have 
also  seen  that  Marmier  succeeded  in  immunising  animals  by 
using  a  toxin  isolated  by  him.  Even,  however,  as  a  method  of 
immunising  animals  for  scientific  observations  Pasteur's  method 
still  obtains. 

Serum  Anticharbonneux.  —  The  properties  of  the  serum  of 
animals  vaccinated  against  anthrax  have  been  investigated  by 
Marchoux.  The  animals  were  immunised  in  the  usual  way. 
The  serum  of  sheep  and  especially  of  rabbits  was  found  to  afford 
a  certain  degree  of  protection  to  susceptible  animals  against 
subsequent  inoculation  with  virulent  bacilli.  It  also  exhibited 


METHODS    OF   EXAMINATION.  317 

a  small  degree  of  curative  action.  When  it  was  injected  im- 
mediately after  inoculation  with  the  bacilli  a  certain  number  of 
the  animals  survived,  but  in  proportion  as  the  symptoms,  of  the 
disease  (oedema,  fever,  etc.)  were  established,  so  was  the  curative 
effect  diminished,  even  though  large  doses  of  the  serum  were 
employed.  These  results  have  been  in  the  main  confirmed  by 
other  observers.  The  difficulties  in  the  way  of  the  therapeutic 
use  of  such  sera,  which  aim  at  the  killing  of  infecting  bacteria, 
will  be  discussed  in  connection  with  Immunity :  here  it  need 
only  be  said  that  different  bodies  must  be  present  in  a  serum  in 
order  that  it  may  be  efficacious,  and  if  all  the  factors  are  not 
present,  then  a  serum  may  have  little  or  no  action.  In  this  con- 
nection it  may  be  mentioned  that,  according  to  de  Nittis,  the 
serum  of  a  pigeon  immunised  against  anthrax  will  protect  a 
guinea-pig  against  a  fatal  infection.  The  serum  of  an  immune 
guinea-pig,  on  the  other  hand,  will  not  protect  a  fresh  guinea- 
pig  or  a  mouse  against  such  an  infection. 

Methods  of   Examination.  —  These  include  (a)  microscopic 
•examination;  (^)the  making  of  cultures  ;  and (c)  test  inoculations. 

(a)  Microscopic  Examination.  —  In  a  case  of  suspected  malig- 
nant pustule,  film  preparations  should  be  made  from  the  fluid 
in  the  vesicles  or  from   a  scraping  of  the  incised  or  excised 
pustule,  and  stained  with  a  watery  solution  of  methylene-blue 
and  also  by  Gram's  method.     By  this  method  practically  con- 
clusive evidence  may  be  obtained  ;  but  sometimes  the  result 
is  doubtful,  as  the  bacilli  may  be  very  few  in  number.     In  all 
cases    confirmatory   evidence    should    be    obtained    by  culture. 
Occasionally  they  are  so  scanty  that  both  film  preparations  made 
from  different  parts  and  even  cultures  may  give  negative  results, 
and  yet  a  few  bacilli  may  be  found  when  a  section  of  the  pustule 
is  examined.     It  should  be  noted  that  the  greatest  care  ought  to 
be  taken  in  manipulating  a  pustule  before  excision,  as  the  diffu- 
sion of  the  bacilli,  into  the  surrounding  tissues  may  be  aided  and 
the  condition  greatly  aggravated.    The  examination  of  the  blood 
in  cases  of  anthrax  in  man  usually  gives  negative  results,  with 
the  exception  of  very  severe  cases,  when  a  few  bacilli  may  be 
found  in  the  blood  shortly  before  death,  though  even  then  they 
may  be  absent. 

(b)  Cultivation.  —  A  small  quantity  of  the  material  used  for 
microscopic  examination  should  be  taken  on  a  platinum  needle, 


318  ANTHRAX. 

and  successive  strokes  made  on  agar  tubes,  which  are  then 
incubated  at  37°  C.  At  the  end  of  twenty-four  hours  anthrax 
colonies  will  appear,  and  can  be  readily  recognised  from  their 
wavy  margins,  by  means  of  a  hand  lens.  They  should  also  be 
examined  microscopically  by  means  of  film  preparations. 

(c)  Test  Inoculations.  —  A  little  of  the  suspected  material 
should  be  mixed  with  some  sterile  bouillon  or  water,  and  injected 
subcutaneously  into  a  guinea-pig  or  mouse,  or  it  may  be  intro- 
duced into  the  subcutaneous  tissue  by  means  of  a  seton.  If 
anthrax  bacilli  are  present,  the  animal  usually  dies  within  two- 
days,  with  the  changes  in  internal  organs  already  described. 


CHAPTER   XV. 

TYPHOID  FEVER  — BACILLI   ALLIED  TO   THE   TYPHOID 
BACILLUS. 

OTHER  NAMES:  —  ENTERIC  FEVER:  GASTRIC  FEVER.  GERMAN, 
TYPHUS  ABDOMINALIS  :  ABDOMINALTYPHUS  :  UNTERLEIBS- 
TYPHUS.  FRENCH,  LA  FIEVRE  TYPHOIDE. 

Historical  Summary. — The  first  definite  descriptions  of  what 
is  now  know  as  the  bacillus  typhosus  appeared  about  1 880-81, 
when  it  was  described  by  Eberth,  Koch,  and  Klebs ;  and  on  ac- 
count of  priority  of  publication  by  the  first-named  observer  it  is 
often  called  Eberth's  bacillus.  Eberth  in  certain  cases  of  the 
disease  found  on  microscopic  examination  characteristic  bacilli 
in  the  intestinal  ulcers,  in  the  spleen,  and  in  the  lymphatic 
glands,  but  made  no  attempts  to  grow  them  outside  the  body. 
Gaffky  (1884)  confirmed  Eberth's  observations  and  obtained 
from  the  spleen  pure  cultures  in  gelatin.  He  further  described 
very  fully  the  morphological  character  of  the  bacilli,  and  held 
that  the  bacilli  were  not  putrefactive,  as  they  did-  not  produce 
putrefactive  effects  on  artificial  media ;  but  all  his  attempts  to 
reproduce  by  their  means  the  disease  in  different  species  of 
animals  (including  monkeys)  were  unsuccessful.  The  position, 
therefore,  was  that  in  the  great  majority  of  cases  of  typhoid 
fever,  characteristic  bacilli  could  be  found  and  isolated  in  pure 
culture,  but  that  these  did  not  give  rise  to  the  disease  in  animals. 

The  question  of  the  significance  of  the  occurrence  of  the  B.  typhosus  was 
complicated  when,  in  1885,  Escherich,  working  on  the  first  appearance  of  or- 
ganisms in  the  bowel  of  the  new-born  infant,  described  a  bacillus  which  he 
named  the  bacillus  coli  communis,  sometimes  called  Escherich's  bacillus. 
This  also  was  shown  to  be  identical  with  the  bacillus  neapolitanus  which 
Emmerich  found  in  the  intestines  of  the  victims  of  a  cholera  epidemic  at 
Naples.  Weisser,  who  worked  at  the  subject,  pointed  out  that  the  B.  coli  was 
a  normal  inhabitant  of  the  human  intestine  ;  and,  further,  comparing  the  growth 
characters  of  this  bacillus  with  those  of  the  typhoid  bacillus,  noted  the  simi- 
larities which  exist  between  the  two  microbes.  From  this  time  forward,  the 


320 


TYPHOID   FEVER. 


question  of  the  morphological  relationships  of  the  two  organisms  has  played 
an  important  part  in  the  bacteriological  investigation  of  the  subject.  There 
has  been  much  controversy  as  to  whether  they  are  varieties  of  the  same  species, 
the  result  of  which  is  a  growing  conviction  that  the  two  are  really  distinct. 

The  Bacillus  Typhosus.  —  Microscopic  Appearances.  —  It  is 
sometimes  difficult  to  find  the  typhoid  bacilli  in  the  organs  of 
a  typhoid  patient.  Numerous  sections  of  different  parts  of  a 
spleen,  for  example,  may  be  examined  before  a  characteristic 
group  is  found.  The  best  tissues  for  examination  are  a  Peyer's 
patch  where  ulceration  has  not  yet  commenced  or  where  it  is 
just  commencing,  the  spleen,  the  liver,  or  a  mesenteric  gland. 
The  spleen  and  liver  are  better  than  the  other  tissues  named,  as 
in  the  latter  the  presence  of  the  B.  coli  is  more  frequent.  From 
scrapings  of  such  solid  organs  dried  films  may  be  prepared  and 
stained  for  a  few  minutes  in  the  cold  by  any  of  the  strong  stain- 
ing solutions,  e.g.  with  carbol-thionin-blue,  or  with  Ziehl-Neelsen's 
carbol-fuchsin  diluted  with  five  parts  of  distilled  water.  As  a 
rule  decolorising  is  not  necessary.  For  the  proper  observation 
of  the  arrangement  of  the  bacilli  in  the  tissues,  paraffin  sections 
should  be  prepared  and  stained  in  carbol-thionin-blue  for  a  few 

minutes,  or  in  LofHer's  methy- 
lene-blue  for  one  or  two  hours. 
The  bacilli  take  up  the  stain 
somewhat  slowly,  and  as  they 
are  also  easily  decolorised,  the 
aniline-oil  method  of  dehydra- 
tion maybe  used  with  advantage 
(vide^.  100).  In  such  prepara- 
tions the  characteristic  appear- 
ance to  be  looked  for  is  the 
occurrence  of  groups  of  bacilli 
^  lying  between  the  cells  of  the 

FIG.  113.  — A  specially  large  clump  of  tissue    (Fig.    in).     The   indi- 

typhoid  bacilli  in  a  spleen.     The  individual  -IT 

bacilli  are  only  seen  at  the  periphery  of  the  Vldual  bacilli  are  2  p  to  4  //-  long, 

mass.     (In  this  spleen  enormous  numbers  of  wjth    somewhat    OVal   ends,    and 

typhoid   bacilli  were   shown  by  cultures   to 

be  present  in  a  practically  pure  condition.)  -5    P   in    thickness.       Sometimes 

Paraffin  section;  stained  with  carbol-thionin-  filaments  8  JJL  to   IO  //,  long   may 

blue.     X  500. 

be  observed,   though  they  are 

less  common  than  in  cultures.  It  is  evident  that  one  of  the 
short  oval  forms  may  frequently  in  a  section  be  viewed  end- 


-  **    X        fV'yWit.Y,tf   ^ 

•«  x JS?f*i  <S^-  ; 


ISOLATION   AND    CULTURE   CHARACTERS.  321 

wise,  in  which  case  the  appearance  will  be  circular.  This 
appearance  accounts  for  some,  at  least,  of  the  coccus-like  forms 
which  have  been  described.  The  bacilli  are  decolorised  by 
Gram's  method. 

Isolation  and  Appearances  of  Cultures.  — To  grow  the  organism 
artificially  it  is  best  to  isolate  it  from  the  spleen  or  gall-bladder, 
as  it  exists  there  in  greater  numbers  than  in  the  other  organs,  and 
may  be  the  sole  organism  present  even  some  time  after  death. 
The  spleen  is  removed  whole,  and  a  portion  of  its  capsule  is 
seared  with  a  cautery  to  destroy  all  superficial  contaminating 
organisms.  A  small  incision  is  ^^  jF  %  w^. 

made    into    the    organ  with  a  -v       *S/  *4^/^'// 

sterile  knife,  a  little  of  the  pulp          >*»""*  t      **>jifr*  & 

JklZr^         &    A>A     \  *   *^^^l    ». 

removed  by  a  platinum  needle,          -*i*  \    *  «^y  /O*lf$  ' 

and  agar  or  gelatin  plates  are    .*  Xv»^b^  ^  *V    jk*jSj^"Jl-^' 
prepared,  or  successive  strokes   *-_; 
made  on  agar  tubes.     In  like 

«*  '  £     *^ 

manner  the  gall-bladder  is 
seared  and  punctured  and  cul- 
tures made  from  the  bile.  On 
the  agar  media  the  growths  are 
visible  after  twenty-four  hours' 

incubation    at    37°  C.       On  agar        FIG.  114.  — Typhoid  bacilli;  from  a  young 

.         culture  on  agar,  showing  some  filamentous 
plates    the     Superficial     Colonies    forms.    Stained  with   weak   carbol-fuchsin. 

appear   as  circular  spots,  dull   x  I00°- 

white  by  reflected  light,  bluish-grey  by  transmitted  light. 
Colonies  in  the  substance  of  the  agar  are  small,  and  appear 
as  minute  round  points.  When  viewed  under  a  low  objective, 
the  surface  colonies  are  found  to  be  very  transparent  (requir- 
ing a  small  diaphragm  for  their  definition),  finely  granular  in 
appearance,  and  with  a  very  coarsely  crenated  and  well-defined 
margin.  The  deep  colonies  are  usually  spherical,  sometimes 
lenticular  in  shape,  and  are  smooth  or  finely  granular  on 
the  surface,  and  more  opaque  than  the  superficial  colonies. 
On  making  cover-glass  preparations,  the  bacilli  are  found  to 
present  the  same  microscopic  appearances  as  are  observed  in 
preparations  from  solid  organs,  except  that  there  may  be  a 
greater  number  of  the  longer  forms  which  may  almost  be 
called  filaments  (Fig.  113).  The  same  is  true  of  films  made 
from  young  gelatin  colonies.  Sometimes  the  diversity  in  the 


322 


TYPHOID   FEVER. 


length  of  the  bacilli  is  such  as  to  throw  doubts  on  the  purity  of 
the  culture.  Its  purity,  of  course,  can  be  readily  tested  by  pre- 
paring plates  from  it  in  the  usual  way.  As  a  general  rule  in  a 

young  (twenty- 
four    to    forty- 

'      -—•  e  •'""-.*'/".'_  -  •  J 

eight  hours  old) 
colony,  grown 
at  a  uniform 
temperature, 
the  bacilli  are 
plump,  and  the 
protoplasm 
stains  uniform- 
ly. In  old  cul- 
tures or  in  cul- 
tures which 
have  been  ex- 
posed to  change 
of  temperature, 
the  protoplasm 
stains  only  in 
parts;  there 
may  be  an  ap- 
pearance of  irregular  vacuolation  either  at  the  centre  or  at  the 
ends  of  the  bacilli.  There  is  no  evidence  that  spore  formation 
occurs  in  the  typhoid  bacillus. 

Motility.  —  In  hanging-drop  preparations  the  bacilli  are  found 
to  be  actively  motile.  The  smaller  forms  have  a  darting  or  roll- 
ing motion,  passing  quickly  across  the  field,  whilst  some  show 
rapid  rotatory  motion.  The  filamentous  forms  have  an  undu- 
lating or  serpentine  motion,  and  move  more  slowly.  Hanging- 
drop  preparations  ought  to  be  made  from  agar  or  broth  cultures 
not  more  than  twenty-four  hours  old.  In  older  cultures  the 
movements  are  less  active. 

Flagella.  —  On  being  stained  by  the  appropriate  methods  (vide 
p.  107)  the  bacilli  are  seen  to  possess  many  long  wavy  flagella 
which  are  attached  all  along  the  sides  and  to  the  ends  (Fig.  115). 
They  are  more  numerous,  longer,  and  more  wavy  than  those  of 
the  B.  coli. 

Characters  of  Cultures.  —  Stab-cultures  in  peptone  gelatin  give 


FlG.  115.  —  Typhoid  bacilli,  from  a  young  culture  on  agar,  showing 
flagella.    Stained  by  Van  Ermengem's  method.     X  1000. 


CHARACTERS  OF  CULTURES. 


323 


a    somewhat     characteristic 

appearance.  On  the  sur- 
face of  the  medium  growth 

spreads  outwards   from   the 

puncture    as  a  thin   film   or 

pellicle,      with      irregularly 

wavy  margin  (Fig.  116,  A). 

It    is    semi-transparent    and 

of     a     bluish-white     colour. 

Ultimately      this        surface 

growth  may  reach   the  wall 

of  the  tube.  Not  infre- 
quently, however,  the  sur- 
face growth  is  not  well 

marked.      Along    the     stab 

there   is   an  opaque  whitish 

line    of    growth,    of    finely 

nodose  appearance.     There 

is    no    liquefaction    of    the 

medium,    and   no   formation 

of   gas.      In  stroke-cultures 

there  is  a  thin  bluish-white 

film,  but  it  does  not  spread 
to  such  an  extent  as  in  the  case  of  the  surface  growth  of  a 
stab-culture  (Fig.  116,  B).     In  gelatin  plates  also  the  superficial 

and  deep  colonies  present  cor- 
responding differences.  The 
former  are  delicate  semi-trans- 
parent films,  with  wavy  margin, 
and  are  much  larger  than  the 
colonies  in  the  substance,  which 
appear  as  small  round  points 
(Fig.  117).  These  appearances, 
which  are  well  seen  on  the  third 
or  fourth  day,  resemble  those 
seen  in  agar  plates,  as  already 
described  in  the  method  of 
FIG.  n7.  — colonies  of  the  typhoid  bacii-  isolation;  but  on  gelatin  the 

lus  (one  superficial  and  three   deep)  in  a    surface      colonies       are       rather 
gelatin  plate.    Three  days'  growth  at  room 

temperature,    x  15.  more  transparent  than  those  on 


FIG.  116. 

A.  Stab-culture  of  the  typhoid  bacillus  in  gelatin, 
five  days'  growth. 

B.  Stroke-culture  of  the  typhoid  bacillus  on  gela- 
tin, six  days'  growth. 

C.  Stab-culture  of  the  bacillus  coli  in  gelatin,  nine 
days'  growth;   the  gelatin   is   split  in    its   lower  part 
owing  to  the  formation  of  gas. 


324  TYPHOID   FEVER. 

agar.  Their  characters,  as  seen  under  a  low  power  of  the 
microscope,  also  correspond. 

In  stroke-cultures  on  agar  there  is  a  bluish-grey  film  of 
growth,  with  fairly  regular  margins,  but  without  any  character- 
istic features.  This  film  is  loosely  attached  to  the  surface,  and 
can  be  easily  scraped  off. 

The  growth  on  potatoes  is  most  important  For  several  days 
(at  ordinary  temperature)  after  inoculation  there  is  apparently 
no  growth.  If  looked  at  obliquely,  the  surface  appears  wet,  and 
if  the  surface  is  scraped  with  the  platinum  loop,  a  glistening 
track  is  left ;  a  cover-glass  preparation  shows  numerous  bacilli. 
Later,  however,  a  slight  pellicle  with  a  dull,  somewhat  velvety 
surface,  may  appear,  and  this  may  even  assume  a  brown  appear- 
ance. These  characteristic  appearances  are  only  seen  when  a 
fresh  potato  with  an  acid  reaction  has  been  used.  In  America, 
at  least,  the  so-called  invisible  growth  upon  potato  which  was  for- 
merly looked  upon  as  the  most  important  means  of  recognition 
has  proven  to  be  a  very  unreliable  test.  For,  on  potatoes  from 
some  sections  of  the  country,  a  growth  quite  like  that  of  B.  coli 
is  more  often  the  rule  than  the  exception.  This  can  possibly  be 
due  either  to  the'  variety  of  the  potato,  or  to  some  variation  in 
its  composition  dependent  upon  the  character  of  the  soil  in  which 
it  grows,  acidity  apparently  having  nothing  to  do  with  the  phe- 
nomena of  visible  or  invisible  growths. 

In  bouillon  incubated  at  37°  C.  for  twenty-four  hours,  there 
is  simply  a  uniform  turbidity.  Cover-glass  preparations  made 
from  such  sometimes  show  filamentous  forms  of  considerable 
length,  without  apparent  segmentation. 

In  litmus  milk  a  slight  degree  of  acidity  is  produced,  causing 
the  milk  to  assume  a  lilac  colour ;  more  rarely,  in  some  instances 
this  acidity  diminishes  and  is  lost,  being  replaced  by  a  strong 
degree  of  alkalinity.  No  coagulation  of  casein  occurs. 

Conditions  of  Growth,  etc.  —  The  optimum  temperature  of 
the  typhoid  bacillus  is  about  37°  C.,  though  it  also  flourishes 
well  at  the  room  temperature.  It  will  not  grow  below  9°  C.  or 
above  42°  C.  Growth  takes  place  in  anaerobic  as  well  as  in 
aerobic  conditions.  Its  powers  of  resistance  correspond  with 
those  of  most  non-sporing  bacteria.  It  is  killed  by  exposure 
for  half  an  hour  at  60°  C.,  or  for  two  or  three  minutes  at 
100°  C.  Typhoid  bacilli  kept  in  distilled  or  ordinary  tap 


BACILLUS    COLI   COMMUNIS.  325 

water  have  usually  been  found  to  be  dead  after   three  weeks 
(Frankland). 

The  Bacillus  coli  communis.  —  This  bacillus  is  the  chief 
organism  present  in  the  small  intestine  in  normal  conditions, 
and,  with  many  other  bacteria,  it  also  inhabits  the  large  intes- 
tine. During  typhoid  fever,  and  other  pathological  conditions 
affecting  the  intestines,  it  is  relatively  and  absolutely  enormously 
increased  in  the  latter  situation,  where  it  may  sometimes  be 
almost  the  only  bacillus  present.  Its  relations  to  various  suppu- 
rative  and  inflammatory  conditions  are  described  in  the  chapter 
on  Suppuration  (p.  196).  Microscopically  it  has  the  same  appear- 
ances and  staining  reaction  as  the  typhoid  bacillus,  and  like  the 
latter  also  presents  variations  in  size,  though  it  is  usually  some- 
what shorter  (Fig.  118).  It  is  usually  sluggishly  motile,  but 
occasionally  motility  seems  to  be  quite  absent,  and  it  possesses 
lateral  flagella,  which,  however,  are  fewer  in  number  and  some- 
what shorter  than  those  of  the  typhoid  bacillus.  It  is  easily 
isolated  from  the  stools  of  men  and  animals  by  any  of  the  ordi- 
nary methods.  After,  e.g.  twenty-four  hours'  incubation  at  37°  C. 
on  agar,  there  are  large  superficial  colonies  and  small  deep 
colonies  in  the  plates.  To  the  naked  eye  they  are  denser  and 
more  glistening  than  those  of 
typhoid  when  viewed  by  trans- 
mitted light,  and  rather  of  a 
brownish-white  colour.  Under 
a  low  objective  the  colonies, 
again,  appear  denser  than  those 
of  the  typhoid  bacillus  and  more 
granular.  On  ordinary  gelatin 
and  agar  media  the  appearances 
are  similar  to  those  of  the 
typhoid  bacillus,  but  the  growth 
is  whiter,  thicker,  and  more 

Opaque,   and    gives    the    impres-         FIG.  n8.  — Bacillus  coli  communis.    Film 
Sion  Of    having    greater   vigour.    Preparation  from  a  young  culture  on  agar. 

Stained  with  weak  carbol-fuchsin.      X  loco. 

In   the    case   of    gelatin    stab- 
cultures  a  few  gas  bubbles  sometimes   develop  in  the  medium 
(Fig.  1 1 6,  C)  due  to  presence  of  muscle  sugar  in  the  beef  infusion. 
On  potatoes  in  forty-eight  hours  there  is  a  distinct  film  of  growth 
of  brownish  tint  and  moist-looking  surface,  which  rapidly  spreads 


326  TYPHOID   FEVER. 

and  becomes  thicker.  This  contrasts  very  markedly  with  the 
colourless  film  of  the  B.  typhosus.  Litmus  milk  assumes 
within  eighteen  hours  a  marked  acidity  as  shown  by  the  red 
colour  of  the  medium,  and  the  milk  is  usually  coagulated  at  any 
period  within  four  days  to  one  month.  Occasionally  some 
varieties  are  met  with  which  actually  fail  to  cause  coagulation. 

The  Comparative  Culture  Reactions  of  the  B.  typhosus  and 
the  B.  coli.  —  The  importance  of  the  relationships  between  the 
B.  typhosus  and  the  B.  coli  has  caused  great  attention  to  be  paid 
to  their  biological  characters,  in  order  to  facilitate  the  distinction 
of  the  one  from  the  other.  Some  of  these  we  have  already  noted. 
Of  the  morphological  characters  the  growth  on  potatoes  is  the 
most  important.  As  has  been  pointed  out  by  Wathelet,  and 
also  by  Klein,  differences  exist  in  the  growth  of  the  two  bacilli 
in  melted  gelatin.  A  gelatin  tube  is  inoculated,  and,  instead  of 
being  kept  at  the  room  temperature,  is  placed  in  the  incubator 
at  37°  C,  at  which  temperature  it  is  of  course  fluid.  In  such 
cultures,  in  the  case  of  the  B.  typhosus,  there  is  a  general 
turbidity  of  the  gelatin,  while  with  the  B.  coli  there  are  large 
flocculi  developed  which  float  on  the  surface.  It  is,  however, 
to  physiological  differences  between  the  bacilli,  rather  than  to 
morphological,  that  importance  is  to  be  attached.  Several 
important  points  are  to  be  studied  hereon. 

(i)  The  Fermentation  of  Sugars.  —  Chantemesse  and  Widal 
were  the  first  to  show  that  the  B.  coli  produced  an  acid  fermen- 
tation in  lactose  (milk  sugar).  Their  method  was  as  follows : 
To  tubes  of  2  per  cent  lactose  bouillon  about  I  gramme  of  steril- 
ised calcium  carbonate  was  added  in  each  case,  and  the  tubes 
were  then  sterilised.  On  inoculating  such  a  tube  with  B.  coli, 
the  acid  produced  by  the  fermentation  (chiefly  lactic  acid)  acts 
on  the  calcium  carbonate,  setting  free  bubbles  of  carbon  dioxide 
which  collect  on  the  surface  of  the  liquid.  The  production  of 
acid  in  lactose  gelatin  by  the  B.  coli  can  also  be  observed  by 
adding  to  tubes  sufficient  blue  litmus  to  make  the  whole  dis- 
tinctly blue.  If  a  stab-culture  be  made  in  such  a  tube,  a  red 
colour  diffuses  out  in  the  gelatin  from  the  line  of  growth,  and 
bubbles  of  gas  also  form.  Later  the  medium  becomes  decolor- 
ised by  reduction  of  the  litmus.  The  addition  of  lactose  01  other 
sugars  to  a  simple  solution  of  peptone,  however,  gives  more 
accurate  results  (p.  80).  The  fermentation  of  lactose  by  B.  coli 


REACTIONS    OF   B.    TYPHOSUS   AND   B.   COLL  327 

may  also  be  demonstrated  by  means  of  Petruschky's  litmus-whey 

(P-  4i). 

The  fermentation  of  sugars  is  a  very  important  effect  of  the 
growth  of  the  B.  coli.1  In  a  culture  on  a  medium  equally  rich 
in  lactose,  for  example,  and  peptone,  the  former  will  be  broken 
up  and  the  latter  be  left  practically  unaffected..  According  to 
the  first  results  of  Chantemesse  and  Widal,  the  B.  typhosus  did 
not  ferment  lactose ;  and  Pere  stated  that  though  it  cannot  fer- 
ment cane  sugar  or  lactose,  it  can  originate  such  a  change -in 
arabinose,  galactose,  levulose,  and  dextrose,  but  with  regard  to 
the  last  this  is  very  doubtful.  Petruschky,  however,  holds  that 
it  can  break  up  lactose  in  litmus-whey.  Much  seems  to  depend 
upon  what  other  constituents  are  present  in  the  medium,  and 
also  on  its  reaction.  The  fermentative  power  of  the  typhoid 
bacillus  is  undoubtedly  much  less  active  than  that  of  the  B.  coli ; 
and  as  a  matter  of  practical  experience  the  formation  of  bubbles 
of  gas  in  Chantemesse  and  Widal's  lactose  medium  is  rarely 
observed.  The  test  may,  therefore,  be  taken  in  conjunction 
with  others,  as  of  use  in  diagnosing  the  identity  of  the  bacillus. 

Dextrose  agar  and  dextrose  gelatin  (2  per  cent)  are  also 
valuable  media.  The  typhoid  bacillus  in  all  cases  produces  no 
gas  in  these  media,  while  with  the  B.  coli  gas  production  is 
observed  in  from  twenty-four  to  forty-eight  hours. 

Curdling  of  Milk  by  the  B.  Coli.  —  This  probably  depends  on 
the  fermentation  of  the  lactose  of  the  milk,  and  the  throwing 
down  of  the  casein  by  the  resulting  lactic  acid ;  but  the  action 
may  be  a  more  complicated  one,  as  milk  can  be  curdled  by 
organisms  which  do  not  possess  acid-forming  properties. 

Formation  of  Acids  in  Ordinary  Media.  —  If  ordinary  litmus 
bouillon  or  gelatin  be  inoculated  with  the  B.  typhosus  or  the  B. 
coli,  a  production  of  acid  will  be  observed  during  the  early  period 
of  growth,  but  the  acid  reaction  is  more  quickly  produced  by 
the  B.  coli. 

With  such  media  Pdre  found  that  in  the  case  of  both  microbes  there  was 
for  forty-eight  hours  a  production  of  acid.  At  the  end  of  five  days,  however, 
typhoid  cultures  were  alkaline,  and  in  cultures  of  B.  coli  the  acidity,  though 
present,  was  diminished.  Ordinary  media  contain  sugars  derived  from  the 

1  For  fuller  information  the  student  is  referred  to  the  valuable  article  of  Pro- 
fessor Theobald  Smith  on  "  The  Fermentation-tube,"  in  the  Wilder  Quarter-Century 
Book,  1893,  P-  l87- 


328  TYPHOID   FEVER. 

meat  from  which  they  are  made,  and  the  acidity  might  proceed' from  the  fer- 
mentation of  these.  In  media  made  with  pure  syntonin  or  peptone,  though 
there  was  an  initial  slight  acid  formation,  especially  with  the  B.  coli,  still  in 
the  case  of  both  organisms  at  the  end  of  four  days  the  reaction  was  alkaline. 
The  reaction  is,  therefore,  probably  a  double  one,  but  the  resulting  acidity  in 
ordinary  cases  may  be  due  to  fermentative  changes  in  carbohydrates.  Here 
again  the  acid-forming  capacities  of  the  B.  typhosus  are  inferior  to  those  of  the 
B.  coli. 

(2)  Prodttction  of  Gas  by  the  B.  coli.  —  The  production  of  gas 
in  various  media  by  the  B.  coli  can  be  demonstrated  by  any  of 
the   methods    described   (p.   78).      Shake-cultures    are    usually 
employed.     According  to  Klein  the  gas  produced  is  methane. 
We  have  found,  however,  that  in  a  shake-culture  in  peptone 
solution  with  10  per  cent  gelatin  added  the  B.  coli  produces  no 
gas,  but  bubbles  rapidly  form  if  the  medium  has  added  to  it  a 
trace  of  lactose.     No  such  development  of  gas  occurs  in  a  shake- 
culture  of  typhoid  in  any  of  these  media. 

(3)  Formation  of  Indol.  — Among  the  bacteria  capable  of  form- 
ing indol  is  to  be  classed  the  B.  coli.     Indol  can  be  recognised 
in  bouillon  cultures  of  the  B.  coli  three  to  four  days  old  by  the 
usual  tests  (vide  p.  80).     As  there  is   no  evidence  that  it  can 
produce  nitrites,  a  small  quantity  of  the  latter  must  be  added. 
The  typhoid  bacillus  rarely  gives  this  reaction  when  growing  in 
ordinary  conditions,  but  on  the  other  hand,  it  appears  that  some 
varieties  of  the  B.  coli  fail  to  produce  it  also.     Peckham,  how- 
ever, has  found  that  if  the  typhoid  bacillus  be  grown  in  peptone 
solution,1  after  a  few  generations  of  three   days  each  it  may 
acquire  the  property  of    producing  indol.      The  formation  of 
indol  by  an  organism  after  the  first  transference  to  peptone 
solution    from  one  of   the    ordinary  media   may,   however,  be 
accepted  as  evidence  in  favour  of  the  organism  not  being  the 

1  Peckham  directs  this  medium  to  be  made  as  follows :  — 

Beef  muscle 225  grms. 

Trypsin 4      » 

Sodium  chloride 5      „ 

Water I  litre 

The  beef  as  old  as  can  be  gotten,  so  as  to  avoid  muscle  sugar  being  present,  is 
chopped  fine  and  put  into  500  c.c.  of  water  and  the  mixture  made  alkaline  with 
sodium  carbonate.  The  vessel  containing  the  mixture  is  placed  upon  a  water  bath  at 
40°  C.  and  the  trypsin  is  added.  Digest  from  one  to  one  and  a  half  hours,  again  ren- 
der alkaline  and  bring  the  mixture  to  the  boil,  strain  through  gauze  and  filter  when 
cold.  The  reaction  of  the  medium  must  be  such  as  to  require  20-30  c.c.  of  a  deci- 
normal  solution  of  caustic  soda  to  bring  it  to  the  neutral  point  of  phenol-phthaleine. 


DIFFERENTIATING   MEDIA.  329 

typhoid  bacillus.  It  is  to  be  noted  here  that  the  presence  of 
lactose  or  dextrose  in  a  medium  prevents  the  production  of  indol 
by  the  B.  coli.  The  indol  reaction  thus  ought  to  be  sought  for 
in  a  sugar-free  medium. 

(4)  Agar  containing  Neutral-red.  —  The  method   here  is  to 
take  sterile  tubes  of  this  medium  (see  p.  42)  and  either  make 
stab  or   shake  cultures  and  incubate  for  twenty-four  hours  at 
37°  C.     In  the  case  of  the  typhoid  bacillus  no  change  in  the 
colour  occurs,  but  in  the  case  of  the  B.  coli  there  is  developed 
a  beautiful  canary  yellow  with  a  greenish  fluorescence.     The 
value  of  the  medium  as  a  means  of   differentiating  the  two 
organisms  is  still  sub  judice.     Fitz  Gerald  and   Dreyer  have 
shown  that  a  very  important  factor  is  the  reaction  of  agar  or 
glucose  agar,  and  consider  the  difference  in  the  effects  of  the 
two  bacteria  only  one  of   degree.     They  state   that   the  best 
results  are  obtained  by  employing  as  a  medium  a  3  per  cent 
lactose  bouillon,  to  which  .5  per  cent  of  a  I  per  cent  watery 
neutral-red  has  been  added. 

(5)  The  Media  of  Capaldi  and  Proskauer.  — The  first  of  these 
is  a  medium  free  of  albumin,  in  which  B.  coli  grows  well  and 
freely  produces  acid,  while  the  typhoid  bacillus  hardly  grows  at 
all,  and  certainly  will  produce  no  change  in  the  reaction.     Its 
composition  is  as    follows  :  — 

Asparagin 0.2    grms. 

Mannite 0.2       „ 

Sodium  chloride     .....  0.02     „ 

Magnesium  sulphate       ....  o.oi      „ 

Calcium  chloride    .....  0.02     „ 

Potassium  monophosphate      .         .         .  0.2       „ 

Water  (distilled)    .....  100.00  c.c. 

The  second  medium  contains  albumin,  and  is  such  that  the  B. 
coli  produces  no  acid,  while  the  typhoid  bacillus  grows  well  and 
produces  an  acid  reaction.  It  consists  of  :  — 

Witte's  peptone      .         .         .         .         -         2.0   grms. 
Mannite          .         .         .         .         .         •         o.i       „ 
Water  (distilled) 100.0  c.c. 

After  the  constituents  of  each  medium  are  mixed  and  dissolved  it 
is  steamed  for  one  and  a  half  hours  and  litmus  solution  added  (the 
tint  being  judged  of  by  experience),  and  the  medium  is  then 


330  TYPHOID    FEVER. 

made  neutral  —  the  first  medium,  being  usually  naturally  acid, 
by  sodium  hydrate,  the  second,  being  usually  alkaline,  by  citric 
acid.  The  medium  is  then  filtered,  filled  into  tubes  containing 
5  c.c.,  and  these  are  sterilised.  After  inoculation  the  character- 
istic appearances  ought  to  manifest  themselves  in  about  twenty 
hours. 

(6)  Growth  on  Phenolated  Media.  —  It  was  at  one  time  thought  the  addi- 
tion of  .2  per  cent  carbolic  acid  to  the  ordinary  media  inhibited  the  growth  of 
all  bacteria  but  the  typhoid  bacillus.     It  has  been  found,  however,  that  the 
growth  of  the  B.  coli  is  also  unaffected  by  such  a  medium,  though  it  prevents 
the  growth  of  most  putrefactive  organisms  which  liquefy  gelatin. 

(7)  The  Application  of  the  Agglutination  Test  in  distinguish- 
ing B.  typhosus  front  B.  coli.  —  The  scope  of  the  application  of 
this  test  will  be  discussed  later  (see  Immunity).     Here  we  may 
say  that  a  negative  result  obtained  with  a  suspected  B.  typhosus 
culture  is  of  greater  value  than  a  positive  result  obtained  with  a 
suspected  B.  coli  culture.     The  test  is  only  to  be  taken  in  con- 
junction with  the  other  means  of  differentiating  the  two  organ- 
isms and  is  not  strictly  a  crucial  one. 

It  will  thus  be  seen  that  the  diagnosis  between  the  B. 
typhosus  and  the  B.  coli  is  a  matter  of  no  small  difficulty.  The 
points  to  be  attended  to  in  making  such  a  diagnosis  have 
been  given  above.  There  is  no  evidence  that  the  one  or- 
ganism ever  passes  into  the  other.  Klein  has  found  that  both 
after  prolonged  sojourn  in  distilled  and  tap  water,  and  also  after 
passage  through  the  bodies  of  a  series  of  animals,  each  organism 
still  preserves  its  original  characters.  Statements  as  to  their 
identity  usually  rest  on  theoretical  considerations,  or  on  purely 
negative  evidence.  Great  difficulties  sometimes  arise  in  con- 
sequence of  a  bacillus  being  found  which,  while  giving  a  number 
of  the  characteristics  of  either  one  or  the  other,  fails  to  give 
some  of  the  characteristic  tests,  or  only  gives  them  very  slowly. 
This  is  especially  true  of  organisms  related  to  the  B.  coli.  It  has 
consequently  become  common  to  speak  of  the  typhoid  group 
and  the  coli  group  in  order  that  such  varieties  may  be  included. 
In  the  coli  group  cases  may  be  met  with  which  do  not  give  an 
indol  reaction  (the  so-called  paracolon  group),  which  do  not 
curdle  milk,  or  which  do  not  produce  gas,  and  Gordon  even 
includes  varieties  producing  alkali,  or  slowly  liquefying  gelatin. 
Three  of  the  most  important  varieties,  the  bacillus  enteritidis 


ALLIED   BACILLI.  331 

(Gaertner),  the  so-called  paracolon  bacillus,  and  the  bacillus  of 
psittacosis  may  be  described. 

Bacillus  Enteritidis  (Gaertner).— In  1888  Gaertner,  in  investigating  a 
number  of  cases  of  illness  resulting  from  eating  the  flesh  of  a  diseased  cow, 
isolated,  not  only  from  the  meat  but  from  the  spleen  of  a  man  who  died,  a 
bacillus  which  presents  all  the  characteristics  of  the  B.  typhosus  except  that  it 
ferments  dextrose  and  is  very  pathogenic  to  animals.  In  the  latter,  whatever 
the  method  of  introduction,  there  is  an  intense  haemorrhagic  enteritis  with 
swelling  of  the  lymph  follicles.  The  distribution  of  the  bacilli  varies  in  different 
cases,  but  usually  they  are  present  in  the  solid  organs.  In  man  also  the  symp- 
toms are  centred  in  the  intestine,  and  hence  the  name  given  to  the  bacillus. 
During  recovery  a  very  characteristic  point  is  the  occurrence  of  desquamation 
of  the  epidermis.  Since  it  was  described  by  Gaertner  others  have  isolated  the 
bacillus  under  similar  circumstances.  Its  toxic  products  have  been  found  to  be 
very  pathogenic  to -animals,  and  in  man  cases  of  illness  have  occurred  when 
broth  made  from  the  diseased  flesh  has  been  partaken  of.  When  there  is  an 
infection  by  the  bacillus  itself,  symptoms  usually  begin  after  twenty-four  hours. 
Many  cases,  however,  of  an  earlier  illness  have  occurred,  no  doubt  due  to  the 
action  of  toxins  already  existing  in  the  meat.  During  the  last  few  years,  in  some 
epidemics  of  meat-poisoning,  similar  bacilli  differing  slightly  from  Gaertner's 
bacillus  have  been  isolated,  e.g.  by  Durham,  and  it  is  probable  that  here  also 
we  have  to  do  not  with  one  variety  but  with  a  group  of  bacilli,  probably  of 
the  same  species  and  possessing  more  or  less  similar  pathogenic  properties. 

The  Paracolon  (Paratyphoid)  Bacillus.  —  Under  the  names  paracolon  or 
paratyphoid  bacillus,  Widal,  Gwyn,  Schottmuller,  and  others  in  Europe  and 
America,  have  described  bacilli  associated  with  continued  fevers,  whose  clinical 
features  were  identical  with  those  of  typhoid  fever,  but  in  none  of  which  could 
the  presence  of  B.  typhosus  be  positively  determined  by  either  blood  reaction 
or  by  cultural  means.  Morphologically,  these  bacilli  resemble  B.  typhosus 
closely,  but  may  be  readily  differentiated  from  it  both  culturally  and  by  serum 
reaction.  They  ferment  glucose,  but  not  lactose  or  saccharose ;  litmus  milk 
at  first  strikes  a  mild  acid  reaction,  with  a  blue-green  cream-ring,  but  about 
the  fourth  or  fifth  day  gradually  loses  this  acidity  and  slowly  becomes  alkaline, 
with  no  coagulative  phenomena;  the  potato  growth  is  usually  like  that  of  B. 
coli,  but  may  be  at  times  almost  invisible ;  the  production  of  indol  is  incon- 
stant. Typhoid  sera  fail  to  agglutinate  paracolon  bacilli,  and  vice  versa, 
paracolon  sera  never  clump  B.  typhosus;  and  further,  the  serum  from  one 
paracolon  infection  may  not  be  able  to  agglutinate  the  bacilli  derived  from 
another.  From  B.  coli  they  are  easily  distinguished  by  their  fermentative 
reactions  and  by  their  behaviour  in  litmus  milk.  Their  position  is  undoubtedly 
in  that  group  designated  by  Durham,  in  his  interesting  study  of  colon  bacillus 
and  allied  forms,  as  the  B.  enteritidis  group. 

The  Psittacosis  Bacillus.  — When  parrots  are  imported  from  the  tropics  in 
large  numbers  many  die  of  a  septicaemic  condition  in  which  an  enteritis,  it  may 
be  haemorrhagic,  is  a  marked  feature.  There  is  intense  congestion  of  all 
the  organs  and  peritoneal  ecchymoses.  From  the  spleen,  bone  marrow,  and 
blood  there  has  been  isolated  a  short  actively  motile  bacillus  with  rounded 


332  TYPHOID   FEVER. 

ends  which  does  not  stain  by  Gram's  method.  //  grows  on  all  ordinary 
media,  and  on  potato  resembles  B.  coli.  It  does  not  liquefy  gelatin,  does 
not  ferment  lactose,  does  not  curdle  milk,  and  gives  no  indol  reaction.  The 
parrot  is  most  susceptible  to  its  action,  but  it  also  causes  a  fatal  haemorrhagic 
septicaemia  in  guinea-pigs,  rabbits,  mice,  pigeons,  and  fowls,  the  bacilli  after 
death  being  chiefly  in  the  solid  organs.  From  affected  parrots  the  disease 
appears  to  be  readily  communicable  to  man,  chiefly,  it  is  probable,  from  the 
feathers  being  soiled  by  infective  excrement.  Several  small  epidemics  have 
been  recognised  and  investigated  in  Paris.  After  about  ten  days'  incubation, 
headache,  fever,  anorexia  occur,  followed  by  great  restlessness,  delirium, 
vomiting,  often  diarrhoea,  and  albuminuria.  Frequently  broncho-pneumonia 
supervenes,  and  a  fatal  result  has  followed  in  about  a  third  of  the  cases 
observed.  The  organism  has  been  isolated  from  the  blood  of  the  heart. 
The  psittacosis  bacillus  is  evidently  one  of  the  typhoid  group,  a  fact  which  is 
further  borne  out  by  the  observation  that  it  is  clumped  by  a  typhoid  serum  — 
i-io  (normal  serum  having  no  result).  The  clumping  is,  however,  said  to 
be  incomplete,  as  the  bacilli  between  the  clumps  may  retain  their  motility. 
It  differs  from  the  typhoid  bacillus  in  its  growth  on  potatoes  and  in  its 
pathogenicity. 

Pathological  Changes  in  Typhoid  Fever.  —  Here  we  confine 
our  attention  solely  to  the  bacteriological  aspects  of  the  disease. 
The  inflammation  and  ulceration  in  the  Peyers  patches  and  soli- 
tary glands  of  tJie  intestines  are  the  central  features.  In  the 
early  stage  there  is  produced  an  acute  inflammatory  condition, 
attended  with  extensive  leucocytic  emigration  and  sometimes  with 
small  haemorrhages.  At  this  period  the  typhoid  bacilli  are  most 
numerous  in  the  patches,  groups  being  easily  found  between  the 
cells.  The  subsequent  necrosis  is  evidently  in  chief  part  the 
result  of  the  action  of  the  toxic  products  of  the  bacilli,  which 
gradually  disappear  from  their  former  positions,  though  they  may 
still  be  found  in  the  deeper  tissues  and  at  the  spreading  margin 
of  the  necrosed  area.  They  also  occur  in  the  lymphatic  spaces 
of  the  muscular  coat.  It  is  to  be  remarked  that  the  number  of 
the  ulcers  arising  in  the  course  of  a  case  bears  no  relation  to  its 
severity.  Small  ulcers  may  occur  in  the  lymphoid  follicles  of 
the  large  intestine.  In  this  regard  some  interesting  observa- 
tions have  of  late  been  made  by  Chiari  and  Krause,  Flexner  and 
Harris,  Lartigau,  and  Ophiils,  wherein  they  state  that  these  typi- 
cal ulcerative  lesions  of  the  intestine  may  be  entirely  wanting, 
(or  so  poorly  developed  as  to  be  overlooked  [Opie  and  Bassett]), 
and  yet  the  bacilli  be  found  in  the  various  organs  or  blood. 

The  mesenteric  glands,  corresponding  to  the  affected  part  of 
the  intestine  are  usually  enlarged,  sometimes  to  a  very  great 


PATHOLOGICAL   CHANGES.  333 

extent,  the  whole  mesentery  being  filled  with  glandular  masses. 
In  such  glands  there  may  be.  acute  inflammation,  and  occasion- 
ally necrosis  in  patches  occurs.  Sometimes  on  section  the  glands 
are  of  a  pale-yellowish  colour,  the  contents  being  diffluent  and 
consisting  largely  of  leucocytes.  Typhoid  bacilli  may  be  isolated 
both  from  the  glands  and  the  lymphatics  connected  with  them, 
but  the  B.  coli  is  in  addition  often  present. 

The  spleen  is  enlarged,  —  on  section  usually  of  a  fairly  firm 
consistence,  of  a  reddish-pink  colour,  and  in  a  state  of  congestion. 
Of  all  the  solid  organs  it  usually  contains  the  bacilli  in  greatest 
numbers.  They  can  be  seen  in  sections,  occurring  in  clumps 
between  the  cells,  there  being  no  evidence  of  local  reaction  round 
them  (Fig.  113).  Similar  clumps  may  occur  in  the  liver  in  any 
situation,  and  without  any  local  reaction.  In  this  organ,  how- 
ever, there  are  often  small  foci  of  leucocytic  infiltration,  in 
which,  so  far  as  our  experience  goes,  bacilli  cannot  be  demon- 
strated. Clumps  of  bacilli  may  also  occur  in  the  kidney. 

In  addition  to  these  local  changes  in  the  solid  organs  there  are  also  wide- 
spread cellular  degenerations  in  the  solid  organs  which  suggest  the  circulation 
of  soluble  poisons  in  the  blood. 

In  the  lungs  there  may  be  bronchitis,  patches  of  congestion  and  of  acute 
broncho-pneumonia.  In  these,  typhoid  bacilli  may  sometimes  be  observed, 
but  evidence  of  a  toxic  action  depressing  the  powers  of  resistance  of  the  lung 
tissue  is  found  in  the  fact  that  the  pneumococcus  is  frequently  found  in  such 
complications  of  typhoid  fever. 

The  studies  of  Voinot  and  of  Nichols  show  that  the  nervous  system  is  often 
seriously  affected  by  marked  alterations  in  the  motor  nerve  cells  of  the  ventral 
horns  of  the  spinal  cord  and  in  the  cells  of  the  posterior  root  ganglia,  with 
extensive  degeneration  of  the  peripheral  nerves  as  well.  Meningitis  and 
brain  abscess  have  been  reported  by  McDaniel  and  McClintock. 

The  blood  in  typhoid  fever  in  probably  80  per  cent  of  all  cases  contains 
the  specific  bacillus,  as  shown  by  the  researches  of  Schottmliller,  Auerbach 
and  Unger,  and  Cole;  at  times  the  bacilli  precede  the  appearance  of  the 
agglutination  phenomenon.  Judging  from  the  results  obtained  by  Karlinski, 
Richardson,  Gwyn,  and  others  B.  typhosus  can  be  isolated  from  the  urine  in  25 
per  cent  of  all  cases  of  the  disease.  That  the  rose-spots  contain  the  bacilli 
seems  undoubtedly  proven  by  the  researches  of  Neufeld,  Curschmann,  and 
Richardson. 

To  sum  up  the  pathology  of  typhoid  fever  we  have  in  it  a 
disease,  the  centre  of  which  lies  in  the  lymphoid  tissue  in  and 
connected  with  the  intestine.  In  this  situation  we  must  have  an 
irritant,  against  which  the  inflammatory  reaction  is  set  up,  and 


334  TYPHOID    FEVER. 

which  in  the  intestine  is  sufficiently  powerful  to  cause  necrosis. 
The  affections  of  the  other  organs  of  the  body  suggest  the  cir- 
culation in  the  blood  of  poisonous  substances  capable  of  depress- 
ing cellular  vitality,  and  producing  histological  changes. 

Suppurations  occurring  in  connection  with  Typhoid  Fever.  — 
With  regard  to  the  relation  of  the  typhoid  bacillus  to  such  condi- 
tions, statements  as  to  its  isolation  from  pus,  etc.,  can  be  accepted 
only  when  all  the  points  available  for  the  diagnosis  of  the  organ- 
ism have  been  attended  to.  On  this  understanding  the  following 
summary  may  be  given.  In  a  small  proportion  of  the  cases  ex- 
amined the  typhoid  bacillus  has  been  the  only  organism  found. 
This  has  been  the  case  in  subcutaneous  abscesses,  in  suppurative 
periostitis,  suppuration  in  the  parotid,  submaxillary,  and  thyroid 
glands,  abscesses  in  the  kidneys,  etc.,  and  probably  also  in  one 
or  two  cases  of  ulcerative  endocarditis.  But  in  the  majority  of 
cases  other  organisms,  especially  the  B.  coli  and  the  pyogenic 
micrococci,  have  been  obtained,  the  typhoid  bacillus  having  been 
searched  for  in  vain.  It  has,  moreover,  been  experimentally 
shown,  notably  by  Dmochowski  and  Janowski,  that  suppuration 
can  be  experimentally  produced  by  injection  in  animals,  espe- 
cially in  rabbits,  of  pure  cultures  of  the  typhoid  bacillus,  the  occur- 
rence of  suppuration  being  favoured  by  conditions  of  depressed 
vitality,  etc.  These  observers  also  found  that  when  typhoid 
bacilli  were  injected  along  with  pyogenic  staphylococci,  the 
former  died  out  in  the  pus  more  quickly  than  the  latter.  Accord- 
ingly, in  clinical  cases  where  the  typhoid  bacillus  is  present 
alone,  it  is  improbable  that  other  organisms  were  present  at  an 
earlier  date. 

Pathogenic  Effects  produced  in  Animals  by  the  Typhoid 
Bacillus.  — There  is  no  disease  known  to  veterinary  science  which 
can  be  said  to  be  identical  with  typhoid,  nor  is  there  any  evidence 
of  the  occurrence  of  the  typhoid  bacillus  under  ordinary  patho- 
logical conditions  in  the  bodies  of  animals.  All  attempts  to 
communicate  the  disease  to  animals  by  feeding  them  on  typhoid 
dejecta  have  been  unsuccessful,  and  though  pathogenic  effects 
have  been  produced  by  introducing  pure  cultures  in  food,  the 
disease  has  borne  no  resemblance  to  human  typhoid.  The 
most  successful  experiments  have  been  those  of  Remlinger,  who 
continuously  fed  rabbits  on  vegetables  soaked  in  water  containing 
typhoid  bacilli.  In  a  certain  proportion  of  animals  symptoms 


EXPERIMENTAL   PATHOLOGY. 


335 


appeared  about  the  sixth  day,  and  the  contamination  of  the  food 
was  then  stopped.  The  illness  which  followed  was  characterised 
by  general  weakness,  diarrhoea,  and  pyrexia  (the  temperature 
curve  being  of  the  nature  of  that  seen  in  human  typhoid),  and  the 
agglutination  reaction  (vide  infra]  was  obtained.  In  some  cases 
recovery  took  place  after  eight  to  twelve  days'  illness  ;  sometimes 
death  after  twelve  to  eighteen  days.  Post  mortem  there  was 
observed  congestion  of  the  small  intestine,  especially  of  the  last 
part,  and  of  Peyer's  patches,  enlargement  of  mesenteric  glands 
and  spleen,  and  in  the  latter  typhoid  bacilli  were  present.  The 
blood  was  sterile.  The  chief  objection  which  can  be  urged  against 
these  experiments  is  that  they  were  performed  in  the  rabbit  — 
an  animal  very  liable  to  be  affected  by  pathogenic  agents  in 
peculiar  ways. 

While  feeding  experiments  are  thus  rather  unsatisfactory, 
the  same  may  be  said  of  the  results  of  subcutaneous  or  intraperi- 
toneal  infection.  Here,  again,  pathogenic  effects  can  easily  be 
produced  by  the  typhoid  bacillus,  but  these  effects  are  of  the 
nature  of  a  short  acute  illness  characterised  by  pyrexia,  rapid 
loss  of  weight,  inability  to  take  food,  and  frequently  ending 
fatally  in  from  twenty-four  to  forty-eight  hours.  The  type  of 
disease  is  thus  very  different  from  what  occurs  naturally  in  man. 
In  such  injection  experiments  the  results  vary  considerably, 
sometimes  scarcely  any  effect  being  produced  by  a  large  dose  of 
a  culture.  This  is  no  doubt  due  to  the  fact  that  different  cases 
of  the  bacillus  vary  much  in  virulence.  Ordinary  laboratory 
cultures  are  often  almost  non-pathogenic.  They  can,  however, 
be  made  virulent  in  various  ways.  This  Chantemesse  and 
Widal  effected  by  injecting  along  with  the  typhoid  culture  the 
sterilised  products  of  the  streptococcus  pyogenes,  and  Sanarelli 
used  for  the  same  purpose  sterilised  cultures  of  the  B.  coli, 
which  were  injected  intraperitoneally  at  the  same  time  as  a 
typhoid  bacillus  was  introduced  subcutaneously.  After  this 
procedure  had  been  repeated  through  a  series  of  animals,  a 
culture  of  typhoid  was  obtained  of  exalted  virulence.  Sidney 
Martin  has  obtained  virulent  cultures  by  passing  bacilli,  derived 
directly  from  the  spleen  of  a  person  dead  of  typhoid  fever, 
through  the  peritoneal  cavities  of  a  series  of  guinea-pigs. 

Sanarelli,  studying  the  effects  of  the  intraperitoneal  injection 
of  a  few  drops  of  a  culture  of  highly  exalted  virulence,  found 


336  TYPHOID   FEVER. 

that  the  Payer's  patches  and  solitary  glands  of  the  intestine 
were  enormously  infiltrated,  sometimes  almost  purulent,  and 
that  they  contained  typhoid  bacilli,  as  also  did  the  mesenteric 
lymphatics  and  glands,  and  the  spleen.  Sanarelli  states  that 
by  whatever  path  the  bacilli  were  introduced  into  the  body,  the 
brunt  of  the  pathological  effects  thus  always  fell  on  the  intes- 
tine and  abdominal  organs.  These  results  are  interesting,  but 
are  not  conclusive  of  the  etiological  relationship  of  the  typhoid 
bacillus  to  human  typhoid.  In  the  latter  it  is  probable  that  the 
pathological  changes  are  due  on  the  one  hand  to  the  direct  local 
reactive  effects  of  the  tissues,  and  on  the  other  to  the  absorption 
of  poisons,  and  therefore  the  artificial  disease  does  not  reproduce 
all  the  incidents  of  that  naturally  arising. 

The  Toxic  Products  of  the  Typhoid  Bacillus.  —  Here  very 
little  light  has  been  thrown  on  the  pathology  of  the  disease,  but 
the  general  results  may  be  outlined.  By  alternately  precipitat- 
ing a  filtered  bouillon  culture  with  alcohol,  and  redissolving  in 
water,  a  toxalbumin  (vide  Chapter  V.)  has  been  obtained  which 
has  pathogenic  effects  of  an  indefinite  kind.  The  toxic  effects 
of  the  bacterial  protoplasm  have  been  investigated  by  Sanarelli, 
who  killed  glycerin  bouillon  cultures  at  60°  C,  and  allowed  the 
bodies  of  the  bacteria  to  macerate.  A  clear  toxic  fluid  could  be 
decanted,  which,  when  injected  subcutaneously,  killed  guinea- 
pigs  in  twenty-four  hours,  with  progressive  fall  of  temperature, 
abdominal  pain,  distention,  and  bloody  stools.  Post  mortem 
there  were  present  peritoneal  exudation,  enlarged  spleen,  con- 
gestion of  the  intestine,  and,  according  to  Sanarelli,  a  special 
infiltration  of  the  lymphoid  patches.  Sidney  Martin  found  that 
the  bodies  of  bacteria  killed  by  chloroform  vapour  were  more 
toxic  (especially  after  being  heated)  than  filtered  cultures. 
Diarrhoea  was  a  constant  symptom  after  injection,  but  no  change 
in  the  Peyerian  patches  was  observed.  Martin  found  that  viru- 
lent cultures  of  the  B.  coli  gave  similar  results  when  similarly 
treated,  and  the  effects  of  both  closely  resemble  those  of  ricin 
and  abrin. 

The  general  result  of  these  and  similar  investigations  is  that 
there  exist  in  the  bodies  of  typhoid  bacilli  toxic  substances,  that 
in  artificial  cultures  do  not  pass  to  any  great  degree  out  into 
the  surrounding  medium,  and  that  though  they  produce  effects 
on  the  intestine,  there  is  evidence  that  such  effects  are  not 


IMMUNISATION   OF   ANIMALS.  337 

peculiar  to  the  toxins  of  the  B.  typhosus.  As  to  the  nature 
of  the  typhoid  toxins,  we  know  nothing.  Martin  has,  however, 
found  that  in  the  case  of  the  typhoid  bacillus  there  is  very  little 
digestive  action,  such  as  occurs  with  the  bacilli  of  diphtheria  and 
tetanus. 

The  Immunisation  of  Animals  against  the  Typhoid  Bacillus. 

In  considering  this  question  we  must  note:  (i)  immunisation 
against  the  living  bacilli;  (2)  immunisation  against  their  tox- 
ins ;  and  (3)  the  relations  between  these  two  conditions.     Earlier 
observers  had  been  successful  in  accustoming  mice  to  the  typhoid 
bacillus  by  the  successive  injections  of   small   and   gradually 
increasing  doses  of  living  cultures  of  the  bacillus.     Later,  Brieger, 
Kitasato,  and  Wassermann  found  that  a  bouillon  made  from  an 
extract  of  the  thymus  gland  contained  bodies  which  were  inimical 
to  the  virulence  of  the  typhoid  bacillus,  though  the  medium  was 
sufficiently  nutritive  to  permit  of  their  multiplication.     A  culture 
three  days  old  in  such  a  bouillon,  killed  by  heating  at  60°  C. 
for  fifteen  minutes,  and  injected  into  mice,  was  without  fatal 
effect.     Ten    days  later  it  was  found   that   these   mice   could 
tolerate  an  otherwise  fatal  dose  of  the  original  living  virulent 
culture.     The  experiments  were  repeated  on  guinea-pigs  with 
a  similar  result,  and  it  was   also  found  that  the  serum  of   a 
guinea-pig   thus   immunised    could,    if    transferred   to   another 
guinea-pig,  protect  the  latter  from  the  subsequent  injection  of 
a  dose  of  typhoid  bacilli  to  which  it  would  naturally  succumb. 
Chantemesse  and  Widal,  Sanarelli,  and  also  Pfeiffer,  succeeded 
in  immunising  guinea-pigs  against  the  subsequent  intraperito- 
neal  injection  of  virulent  living  typhoid  bacilli,  by  repeated  and 
gradually  increasing  intraperitoneal  or  subcutaneous  doses  of 
typhoid  cultures  in  bouillon,  in  which  the  bacilli  had  been  killed 
by  heat  or  chloroform  vapour.      Experiments  performed  with 
serum  derived  from  typhoid  patients  and  convalescents  indicate 
that  similar  effects  occur  in  those  who  have  successfully  resisted 
the  natural  disease.     Thus  many  observers  had  noticed  that  the 
serum  of  men  convalescent  from  typhoid  had  an  inimical  effect 
on  typhoid  bacilli.     Pfeiffer  found  that  the  serum  of  healthy  men 
might  have  such  an  action  but  in  a  much  less  degree.      The 
method  was  to  mix  the  serum  and  the  bacilli  in  a  little  bouillon, 
and  inject  the  whole  intraperitoneally  into  guinea-pigs.      He 
found  that  when  the    latter   did   not   die,   the   bacilli   became 


338  TYPHOID   FEVER. 

motionless  and  apparently  dead,  and  that  plate-cultures  made 
after  a  time  from  the  exudation  containing  them,  remained 
sterile.  The  serum  of  such  patients  has,  therefore,  antibacterial 
powers,  but  there  is  no  evidence  that  it  contains  any  antitoxic 
bodies  (see  chapter  on  Immunity).  Pfeiffer,  for  example,  found 
that  on  adding  serum  from  typhoid  convalescents  to  the  bodies 
of  typhoid  bacilli  killed  by  heat,  and  injecting  the  mixture  into 
guinea-pigs,  death  took  place  as  in  control  animals  which  had 
received  these  toxic  agents  alone.  Sanarelli  also  found  that 
while  the  injection  of  similar  toxic  fluids  rendered  the  animal 
immune  to  a  certain  dose  of  living  bacilli,  it  still  could  be  killed 
by  a  further  dose  of  the  toxin.  Pfeiffer  found  that  by  using  the 
serum  of  immunised  goats  he  could,  to  a  certain  extent,  protect 
other  animals  against  the  subsequent  injection  of  virulent  living 
typhoid  bacilli.  On  trying  to  use  the  agent  in  a  curative  way, 
i.e.  injecting  it  only  after  the  bacilli  had  begun  to  produce  their 
effects,  he  got  little  or  no  result. 

There  is  thus  evidence  that  the  serum  of  persons  who  have 
recovered  from  typhoid  fever,  and  the  serum  of  animals  artificially 
immunised  against  virulent  typhoid  bacilli,  protect  from  these 
bacilli.  There  is  no  evidence  that  the  serum  has  much  power 
in  neutralising  the  intracellular  toxins  of  these  bacilli.  We 
have  thus  this  very  important  fact.  Animals  are  immunised  by 
injections  of-  the  toxins  of  a  bacillus  ;  their  serum,  however,  has 
no  effect  in  neutralising  its  toxins,  but  only  aids  in  the  destruc- 
tion of  the  bacilli  which  produce  the  toxins.  Similar  results 
have  been  obtained  in  the  case  of  cholera. 

The  Pathogenicity  of  the  B.  coli  and  its  Relation  to  that  of  the  Typhoid 
Bacillus. — We  have  already  seen  that  the  B.  coli  is  probably  responsible  for 
the  occurrence  of  some  of  the  abscesses  which  follow  typhoid  fever.  It  is 
also  apparently  the  cause  of  some  cases  of  summer  diarrhoea  (cholera  nostras), 
and  of  infantile  diarrhoea.  Its  numbers  in  the  intestine  are  greatly  increased 
during  typhoid  fever,  and  also  during  any  pathological  condition  affecting 
the  intestine.  Intraperitoneal  injection  in  guinea-pigs  is  occasionally  fatal. 
Subcutaneous  injection  results  in  local  abscesses,  and  sometimes  in  death 
from  cachexia.  Sanarelli  found  that  the  B.  coli  isolated  from  typhoid  stools 
was  much  more  virulent  than  when  isolated  from  the  stools  of  healthy 
persons.  He  holds  that  the  increase  in  virulence  is  due  to  the  effect  of  the 
typhoid  toxins,  and  devised  an  ingenious  experiment  which  seems  to  prove 
this  point.  This  increased  virulence  of  the  B.  coli  in  the  typhoid  intestine 
makes  it  possible  that  some  of  the  pathological  changes  in  typhoid  may  be  due, 
not  to  the  typhoid  bacillus,  but  to  the  B.  coli.  Some  of  the  general  symptoms 


RELATION    OF   B.   TYPHOSUS    TO    TYPHOID.  339 

may  be  intensified  by  the  absorption  of  toxic  products  formed  by  it  and  by  other 
organisms.  It  is  to  be  noted  that  lesions  produced  in  guinea-pigs  are  very 
similar  to  those  of  the  B.  typhosus.  Differences  of  behaviour  of  the  two 
bacilli,  in  connection  with  their  pathological  effects,  have  been  brought  for- 
ward as  confirmatory  of  the  fact  of  their  being  distinct  species.  Thus  Sanarelli 
accustomed  the  intestinal  mucous  membrane  of  guinea-pigs  to  toxins  derived 
from  an  old  culture  of  the  B.  coli,  by  introducing  day  by  day  small  quantities 
of  the  latter  into  the  stomach.  When  a  relatively  large  dose  could  be  tolerated, 
it  was  found  that  the  introduction  in  the  same  way  of  a  small  quantity  of  typhoid 
toxin  was  followed  by  fatal  result.  Pfeiffer  also  found  that  while  the  serum  of 
convalescents  from  typhoid  paralysed  the  typhoid  bacilli,  it  had  no  more  effect 
on  similar  numbers  of  B.  coli  than  the  serum  of  healthy  men. 

General  View  of  the  Relationship  of  the  B.  typhosus  to 
Typhoid  Fever.  —  i.  We  have  in  typhoid  fever  a  disease  having 
its  centre  in  and  about  the  intestine,  and  acting  secondarily  on 
many  other  parts  of  the  body.  In  the  parts  most  affected  there 
is  always  a  bacillus  present,  microscopically  resembling  other 
bacilli,  especially  the  B.  coli,  which  is  a  normal  inhabitant  of  the 
animal  intestine.  This  bacillus  can  be  isolated  from  the  charac- 
teristic lesions  of  the  disease  and  from  other  parts  of  the  body 
as  described,  and  further,  it  is  found  by  culture  reactions  to 
differ  from  the  B.  coli.  The  whole  series  of  culture  reactions, 
however,  must  be  investigated  before  a  particular  bacillus  is 
identified  as  the  B.  typhosus,  and  no  weight  must  be  attached 
to  any  observations  made  on  the  subject  when  this  has  not  been 
done.  Here  the  important  point,  however,  is  that  a  bacillus 
giving  all  the  reactions  of  the  typhoid  bacillus  has  never  been 
isolated  except  from  cases  of  typhoid  fever,  or  under  circum- 
stances that  make  it  possible  for  the  bacillus  in  question  to  have 
been  derived  from  a  case  of  typhoid  fever.  There  is  no  evidence 
that  the  B.  coli  can  be  transformed  into  the  typhoid  bacillus,  or 
the  typhoid  bacillus  into  the  B.  coli,  though  of  course  this  does 
not  preclude  the  possibility  of  the  one  having  been  originally 
derived  from  the  other.  All  practically  are  now  agreed  that 
two  separate  bacilli  exist,  the  B.  coli  and  the  B.  typhosus. 

2.  Against  the  etiological  relationship  of  the  latter  to  the 
disease  several  facts  may  be  adduced.  First,  there  is  the  com- 
parative difficulty  of  the  isolation  of  the  B.  typhosus  from  the 
stools  of  typhoid  patients.  We  have  pointed  out,  however,  that 
the  latter  can  be  isolated  during  the  first  ten  days  of  the  disease, 
and  that  the  extraordinary  multiplication  of  the  B.  coli,  which 
takes  place  in  any  pathological  condition  of  the  intestine, 


340  TYPHOID    FEVER. 

sufficiently  explains  the  failures  in  the  later  stages.  The  second 
and  great  difficulty  in  the  way  of  accepting  the  etiological 
relationship  of  the  B.  typhosus  lies  in  the  comparative  failure  to 
cause  the  disease  in  animals.  We  have  noted,  however,  that  in 
nature  animals  do  not  suffer  from  typhoid  fever. 

3.  The  observations  of  Pfeiffer  and  others  on  the  protective 
power  against  typhoid  bacilli  shown,  on  testing  in  animals,  to 
belong  to  the  serum  of  typhoid  patients  and  convalescents,  and 
the  peculiar  action  of  such  serum  in  immobilising  and  causing 
clumping  of  the  bacilli  (vide  infra)  are  also  of  great  importance. 
Additional  important  evidence  of  the  typhoid  bacillus  being  the 
cause  of  typhoid  fever  is  found  in  the  fact  that  vaccination  by 
means  of  the  dead  bacilli  (vide  infra)  has  a  marked  effect  in 
preventing  the  disease  arising  in  a  protected  population  exposed 
to  infection,  and  also  in  lowering  the  mortality  when  the  fever 
attacks  those  who  have  been  inoculated.  These  facts  may  thus 
be  accepted  as  indirect  but  practically  conclusive  evidence  of  the 
pathogenic  relationships  of  the  typhoid  bacillus  to  the  disease. 

According  to  our  present  results  we  must  thus  hold  that  the 
bacillus  typhosus  constitutes  a  distinct  species  of  bacterium,  and 
that  there  is  every  reason  for  accepting  it  as  the  cause  of  typhoid 
fever.  Evidence  of  an  important  nature  confirmatory  of  this 
view  is,  we  think,  found  in  the  fact  that  cases  have  occurred 
where  bacteriologists  have  accidentally  infected  themselves  by 
the  mouth  with  pure  cultures  of  the  typhoid  bacillus,  and  after 
the  usual  incubation  period  have  developed  typhoid  fever. 
Several  cases  of  this  kind  have  been  brought  to  our  notice  and 
are  not,  we  think,  vitiated  by  the  fact  that  other  similar  instances 
have  occurred  without  the  subsequent  development  of  illness. 
These  latter  would  be  accounted  for  by  a  low  degree  of  suscepti- 
bility on  the  part  of  the  individual  or  to  a  want  of  pathogenicity 
in  the  cultures. 

The  Serum  Diagnosis  of  Typhoid  Fever.  —  This  method  of 
diagnosis  is  based  on  the  fact  that  living  and  actively  motile 
typhoid  bacilli,  if  placed  in  the  diluted  serum  of  a  patient  suffer- 
ing from  typhoid  fever,  within  a  very  short  time  lose  their 
motility  and  become  aggregated  into  clumps.  The  researches 
which  led  up  to  the  discovery  will  be  described  in  the  chapter 
on  Immunity.  We  shall  find  that  in  many  diseases  the  serum 
has  this  property  of  causing  agglutination  of  cultures  of  the 


SERUM    DIAGNOSIS    OF   TYPHOID   FEVER.  341 

causal  bacterium.  The  principles  on  which  the  possession  of 
the  faculty  depends,  and  also  its  significance,  are  obscure,  and 
even  in  the  case  of  the  typhoid  bacillus,  where  an  enormous 
amount  of  work  has  been  done,  we  do  not  know  the  true  inter- 
pretation of  some  of  the  facts  which  have  been  observed. 

The  methods  by  which  the  test  can  be  applied  have  already 
been  described  (p.  109). 

(1)  It  will  be  there  seen  that  the  loss  of  motility  and  clumping 
may  be  observed  microscopically.     If  a  preparation  be  made  by 
the  method  detailed  (typhoid  serum  in  a  dilution  of,  say,  1-50 
having  been  employed),  and  examined  at  once  under  the  micro- 
scope, the  bacilli  will  usually  be  found  actively  motile,  darting 
about  in  all  directions.     In  a  short  time,  however,  these  move- 
ments gradually  become  slower,  the  bacilli  begin  to  adhere  to  one 
another,  and  ultimately  become  completely  immobile  and  form 
clumps  by  their  aggregation,  so  that  no   longer  are  any  free 
bacilli  noticeable  in  the    preparation.     When  this   occurs  the 
reaction  is  said  to  be  complete.     If  the  clumps  be  watched  still 
longer  a  swelling  up  of  the  bacilli  will  be  observed,  with  a 
granulation  of  the  protoplasm,   so  that  their  forms  can  with 
difficulty  be  recognised.     In  a  preparation  similarly  made  with 
non-typhoid  serum  the  individual  bacilli  can  be  observed  separate 
and  actively  motile  for  many  hours. 

(2)  A  corresponding  reaction  visible  to  the  naked  eye  is 
obtained  by  the  "  sedimentation  test,"  the  method  of  applying 
which  has  also  been  described  (p.  112).     Here  at  the  end  of 
twenty-four  hours  the  bacilli  form  a  mass  like  a  precipitate  at  the 
bottom  of  the  mixture  of  bacterial  emulsion  and  diluted  typhoid 
serum,  while  the  upper  part  remains  clear.     A  similar  prepara- 
tion made  with  normal  serum  shows  a  diffuse  turbidity  at  the  end 
of  twenty-four  hours.    The  test  in  this  form  has  the  disadvantage 
of  taking  longer  time  than  the  microscopic  method,  but  it  is 
useful  as  a  control ;  in  nature  it  is  similar. 

Such  is  what  occurs  in  the  case  of  a  typical  reaction.  There 
are  several  details,  however,  which  require  attention,  and  on  which 
the  value  of  the  method  as  a  means  of  diagnosis  largely  depends. 
The  race  of  typhoid  bacillus  employed  is  important.  All  races  do 
not  give  uniformly  the  same  results,  though  it  is  not  known  on 
what  this  difference  of  susceptibility  depends.  The  bacteriologist 
must,  therefore,  apply  a  process  of  selection  to  the  races  at  his 


342  TYPHOID    FEVER. 

disposal,  with  a  view  to  obtaining  one  which  gives  the  best  result 
in  the  greatest  number  of  undoubted  cases  of  typhoid  fever,  and 
which  gives  as  little  reaction  as  possible  with  normal  sera  or  sera 
derived  from  other  diseases.  This  latter  point  is  important,  as 
some  races  react  very  readily  to  non-typhoid  sera.  Again,  care 
must  be  taken  as  to  the  state  of  the  culture  used.  The  suitability 
of  a  culture  may  be  impaired  by  varying  the  conditions  of  its 
growth.  Continued  growth  of  a  race  in  surroundings  very 
favourable  to  vegetable  activity  makes  it  less  suitable  for  use  in 
the  test,  as  the  bacilli  tend  naturally  to  adhere  in  clumps,  which 
may  be  mistaken  for  those  produced  by  the  reaction.  Wyatt 
Johnson  recommends  that  the  stock  culture  should  be  kept 
growing  on  agar  at  room  temperature  and  maintained  by  agar 
sub-cultures  made  once  a  month.  For  use  in  applying  the  test, 
bouillon  sub-cultures  are  made  and  incubated  for  twenty-four 
hours  at  37°  C.  As  the  reaction  of  the  medium  has  also  an  im- 
portant effect  on  the  sensitiveness  of  a  culture,  he  recommends 
that  such  bouillon  should  first  be  made  neutral  to  phenol-phtha- 
leine,  and  then  have  added  to  it  3  or  4  per  cent  of  normal  hydro- 
chloric acid.  When  these  precautions  are  taken  a  growth  occurs 
which  only  gives  a  uniform  turbidity  in  the  bouillon  without  any 
adhesion  of  the  bacilli  in  masses.  It  is  usually,  however,  quite 
safe  to  use  bouillon  prepared  in  the  ordinary  way.  The  relation 
of  the  dilution  of  the  serum  to  the  occurrence  of  clumping  is 
most  important.  It  has  been  found  that  if  the  degree  of  dilution 
be  too  small  a  non-typhoid  serum  may  cause  clumping.  If  pos- 
sible, observations  should  always  be  made  with  dilutions  of 
i-io,  1-30,  1-50,  i-ioo.  To  speak  generally,  the  more  dilute 
the  serum,  the  longer  time  is  necessary  for  a  complete  reaction. 
Some  typhoid  sera  have,  however,  very  powerful  agglutinating 
properties,  and  may  in  a  comparatively  short  time  produce  a 
reaction  when  diluted  many  hundreds  of  times.  The  conditions 
giving  rise  to  such  sera  are  not  known,  and  the  cases  from 
which  they  are  derived  are  not  necessarily  of  a  severe  type. 
With  highly  diluted  sera  not  only  may  the  reaction  be  delayed 
but  it  may  be  incomplete.  Here,  what  is  usually  seen  is  that  the 
clumps  formed  are  small,  many  bacilli  being  left  free.  These 
latter  may  either  have  been  rendered  motionless  or  they  may 
still  be  motile.  No  diagnosis  is  conclusive  which  is  founded  on 
the  occurrence  of  such  an  incomplete  clumping  alone.  Seeing 


AGGLUTINATION    WITH    DRIED    BLOOD.  343 

that  low  dilutions  sometimes1  give  a  reaction  with  non-typhoid 
sera,  great  discussion  has  taken  place  as  to  what  is  the  minimum 
dilution  at  which,  when  complete  clumping  occurs,  it  may  safely 
be  said  that  the  reaction  is  due  to  the  specific  action  of  a  typhoid 
serum.  The  general  consensus  of  opinion,  with  which  our  own 
experience  agrees,  is  that  when  a  serum  in  a  dilution  of  1-30 
causes  complete  clumping  in  half  an  hour,  it  may  safely  be  said 
that  it  has  been  derived  from  a  case  of  typhoid  fever.  Suspicion 
should  be  entertained  as  to  the  diagnosis  if  a  lower  dilution  is 
required,  or  if  a  longer  time  is  required.1 

TJie  Dried-blood  Method.  — This  method  was  introduced  by 
Wyatt  Johnston  of  Montreal,  and  is  especially  useful  for  routine 
work  in  health  departments,  where  obviously  critical  methods 
cannot  be  readily  employed.  As  practised  by  health  boards, 
the  physician  is  instructed  to  cleanse  the  lobe  of  the  ear  or  the 
tip  of  a  finger  of  the  patient  with  soap  and  water  and  alcohol, 
to  draw  blood  by  pricking  the  part  with  a  needle  and  permitting 
a  drop  or  two  of  blood  to  be  deposited  separately  upon  a  pro- 
vided sterile  glass  slide  or  aluminium  strip  and  allowing  the 
blood  to  dry  (in  lieu  of  glass  or  aluminium,  non-absorbent  paper 
may  be  used).  Upon  reaching  the  laboratory  the  sample  is 
covered  by  approximately  five  times  its  amount  of  water  and 
allowed  to  stand  two  minutes,  then  one  loopful  is  removed  to  a 
cover-slip  and  to  it  is  added  one  loopful  of  the  preparation  of 
typhoid  bacilli,  and  the  whole  is  treated  as  a  hanging  drop  in 
the  manner  carried  out  in  quantitative  examination. 

The  reaction  given  by  the  serum  in  typhoid  fever  usually 
begins  to  be  observed  about  the  seventh  day  of  the  disease, 
though  occasionally  it  has  been  found  as  early  as  the  fifth  day, 
and  sometimes  it  does  not  appear  till  the  third  week  or  later. 
Usually  it  gradually  becomes  more  marked  as  the  disease  ad- 
vances, and  it  is  still  given  by  the  blood  of  convalescents  from 
typhoid,  but  cases  occur  in  which  it  may  permanently  disappear 
before  convalescence  sets  in.  How  long  it  lasts  after  the  end  of 
the  disease  has  not  yet  been  fully  determined,  but  in  many  cases 
it  has  been  found  after  several  months  at  least.  As  a  rule,  up 
to  a  certain  point,  the  reaction  is  more  marked  where  the  fever 
is  of  a  pronounced  character,  whilst  in  the  milder  cases  it  is  less 

1  By  American  observers  it  is  usually  conceded  that  a  diagnosis  may  be  made, 
using  a  dilution  of  1-50,  with  a  time  limit  of  two  hours,  without  falling  into  error. 


344  TYPHOID   FEVER. 

pronounced.  In  certain  grave  cases,  however,  the  reaction  has 
been  found  to  be  feeble  or  almost  absent,  and  accordingly  some 
hold  that  a  feeble  reaction  when  the  disease  is  manifestly  severe 
is  of  bad  omen.  In  some  cases,  which  from  the  clinical  symp- 
toms were  almost  certainly  typhoid,  the  reaction  has  apparently 
been  found  to  be  absent. 

It  has  been  found  that  the  reaction  is  not  only  obtained  with 
living  bacilli,  but  in  certain  circumstances  also  with  bacilli  that 
have  been  killed.  This  last  may  be  effected  by  keeping  the 
bacilli  at  60°  C.  for  an  hour.  If  a  higher  temperature  be  used, 
sensitiveness  to  agglutination  is  impaired.  The  capacity  is  also 
still  retained  if  a  germicide  be  employed.  Here  Widal  recom- 
mends the  addition  of  one  drop  of  formalin  to  150  drops  of  cul- 
ture. The  reaction,  however,  tends  to  be  less  complete.  It 
may  be  remarked  that  while  clumping  is  taking  place  where 
dead  cultures  are  used,  active  brownian  movements  among  the 
free  bacteria  may  be  noticed,  which  may  lead  the  observer  to 
doubt  whether  the  bacilli  are  really  dead. 

Besides  the  blood  serum  it  has  been  found  that  the  reaction 
is  given  in  cases  of  typhoid  fever  by  pericardial  and  pleural 
effusions,  by  the  bile  and  by  the  milk,  and  also  to  a  slight 
degree  by  the  urine.  The  blood  of  a  foetus  may  have  little 
agglutinating  effect,  though  that  of  its  mother  may  have  given 
a  well-marked  reaction ;  sometimes,  however,  the  foetal  blood 
gives  a  well-marked  reaction.  It  may  here  also  be  mentioned 
that  a  serum  will  stand  exposure  for  an  hour  at  58°  C.  without 
having  its  agglutinating  power  much  diminished.  Higher  tem- 
peratures, however,  cause  the  property  to  be  lost. 

The  Agglutination  of  Organisms  other  than  the  B.  typhosus 
by  Typhoid  Serum.  —  It  was  at  first  thought  that  reaction  in 
typhoid  fever  would  afford  a  reliable  method  of  distinguishing 
the  typhoid  bacillus  from  the  B.  coli.  Though  many  races  of 
the  latter  give  no  reaction  with  a  typhoid  serum,  there  are  others 
which  react  positively.  Usually,  however,  a  lower  dilution  and 
a  longer  time  are  required  for  a  result  to  be  obtained,  and  the 
reaction  is  often  incomplete.  It  has  also  been  found  that  other 
organisms  belonging  to  the  .typhoid  group,  e.g.  Gaertner's 
bacillus  and  perhaps  the  bacillus  of  psittacosis,  react  in  a  similar 
way.  The  reaction  as  a  method  of  distinguishing  between  these 
forms  is  thus  not  reliable,  but  in  certain  cases  it  may  be  of  value 


AGGLUTINATION    OF   OTHER   BACILLI.  345 

in  giving  confirmation  to  other  tests.  There  is  a  point  in  this 
connection  regarding  which  further  light  is  required.  Many 
races  of  B.  coli  in  use  have  been  isolated  from  typhoid  cases, 
and  we  as  yet  do  not  know  what  effect  a  sojourn  in  such  cir- 
cumstances may  have  on  its  subsequent  sensitiveness  to  agglu- 
tination by  typhoid  serum. 

The  discovery  that  the  exhibition  of  agglutination  is  not 
confined  to  the  B.  typhosus  has  caused  great  attention  to  be 
paid  to  the  sensitiveness  to  different  sera  shown  by  it  and  Uy 
other  allied  organisms.  It  has  been  found  that  not  only  typhoid 
sera  but  the  sera  of  healthy  persons,  and  of  those  suffering  from 
diseases  other  than  typhoid  fever,  may  occasionally  clump 
typhoid  bacilli  even  when  considerably  diluted.  It  has  not, 
however,  been  sufficiently  noted  that,  as  Christophers  has 
pointed  out,  a  large  proportion  of  similar  sera  will  clump  the 
B.  coli  in  dilutions  of  from  1-20  to  1-200,  and  no  doubt  many 
of  the  reactions  shown  by  typhoid  sera  towards  B.  coli  are  due 
to  the  pre-existence  in  the  individuals  of  an  agglutinative  prop- 
erty towards  the  bacillus.  It  has  been  shown  that  both  the 
B.  coli  and  the  B.  typhosus  may  be  clumped  by  the  normal 
serum  of  the  horse,  the  ass,  and  the  rabbit,  and  it  has  been 
found  that  the  serum  of  an  animal  immunised  against  either  of 
these  bacilli  sometimes  clumps  both,  and  sometimes  also  in  ad- 
dition the  B.  enteritidis,  though  usually  the  dilutions  necessary 
differ.  It  may  also  be  remarked  that  in  such  immunised  ani- 
mals the  best  agglutinating  result  is  usually  obtained  with  sub- 
cultures of  the  race  by  which  immunisation  was  effected. 

With  regard  to  the  value  of  the  serum  reaction  there  is  little 
doubt.  In  nearly  95  per  cent  of  cases  of  typhoid  it  can  be 
obtained  in  such  a  form  that  no  difficulty  is  experienced  if  the 
precautions  detailed  above  are  observed.  The  causes  of  pos- 
sible error  may  be  summarised  as  follows :  the  serum  of  the 
person  may  naturally  have  the  capacity  of  clumping  typhoid 
bacilli;  there  may  have  been  an  attack  of  typhoid  fever  pre- 
viously with  persistence  of  agglutinative  capacity  ;  the  case  may 
be  one  of  disease  caused  by  an  allied  bacillus ;  the  disease  may 
have  a  quite  different  cause,  r  d  yet  the  serum  may  react  with 
typhoid  •  bacilli ;  the  disease  may  be  typhoid  fever  and  yet  no 
reaction  may  occur.  The  most  important  of  these  sources  of 
error  is  that  with  which  diseases  caused  by  allied  organisms  are 


346  TYPHOID   FEVER. 

concerned,  as  it  is  probable  that  all  the  forms  which  these  take 
in  man  have  not  been  recognised.  The  very  wide  application 
of  the  reaction  has  elicited  the  fact  that  it  is  given  in  many  cases 
of  slight,  transient,  and  ill-defined  febriculae,  which  occur  espe- 
cially when  typhoid  fever  is  prevalent.  Our  knowledge  of  these 
is  still  insufficient  to  justify  our  setting  all  of  them  down  as  cases 
of  aborted  typhoid.  There  is  no  doubt  that,  taking  all  the  facts 
into  account,  the  cases  where  the  reaction  gives  undoubtedly 
correct  information  so  far  outnumber  those  in  which  an  error 
may  be  made  that  it  must  be  looked  on  as  a  most  valuable  aid 
to  diagnosis.  In  concluding  we  may  say  that  the  fact  of  a 
typhoid  serum  clumping  allied  bacilli  in  no  way,  so  far  as  our 
present  knowledge  goes,  justifies  doubt  being  cast  on  the  specific 
relation  of  the  typhoid  bacillus  to  typhoid  fever. 

Vaccination  against  Typhoid.  —  The  principles  of  the  im- 
munisation of  animals  against  typhoid  bacilli  have  been  applied 
by  Wright  and  Semple  to  man  in  the  following  way.  Typhoid 
bacilli  are  obtained  of  such  virulence  that  a  quarter  of  a  twenty- 
four  'hours'  old  sloped  agar  culture  when  administered  hypo- 
dermically  will  kill  a  guinea-pig  of  from  350  to  400  grammes. 
Vaccination  can  be  accomplished  by  emulsifying  such  a  culture 
in  bouillon,  and  killing  it  by  heating  for  five  minutes  at  60°  C. 
For  use,  from  one-twentieth  to  one-fourth  of  the  dead  culture  is 
injected  hypodermically,  usually  in  the  flank.  The  vaccine  now 
sent  out  by  Wright,  however,  consists  of  a  portion  of  a  bouillon 
culture  similarly  treated.  The  effects  of  the  injection  are  some 
tenderness  locally  and  in  the  adjacent  lymphatic  glands,  and  it 
may  be  local  swelling,  all  of  which  come  on  in  a  few  hours,  and 
may  be  accompanied  by  a  general  feeling  of  restlessness  and  a 
rise  of  temperature,  but  the  illness  is  over  in  twenty-four  hours. 
During  the  next  ten  days  the  blood  of  the  individual  begins  to 
manifest,  when  tested,  a  positive  Widal's  reaction,  and  further, 
Wright  has  found  that  usually  after  the  injection  there  is  a 
marked  increase  in  the  capacity  of  the  blood  serum  to  kill  the 
typhoid  bacilli  in  vitro.  There  is  little  doubt  that  these  observa- 
tions indicate  that  the  vaccinated  person  possesses  a  degree  of 
immunity  against  the  bacillus,  and  this^  conclusion  is  borne  out 
by  the  results  obtained  in  the  use  of  the  vaccine  as  a  prophy- 
lactic against  typhoid  fever.  Extensive  observations  have  been 
made  in  the  British  army  in  India,  and  also  in  the  South  African 


VACCINATION   AGAINST   TYPHOID.  347 

Field  Force,  in  which  the  efficacy  of  the  treatment  was  put  to 
test.  Though  in  isolated  cases  not  much  difference  has  been 
observed  among  those  treated  as  compared  with  those  untreated, 
yet  the  broad  general  result  may  be  said  to  leave  little  doubt 
that  on  the  one  hand  protective  inoculation  diminishes  the 
tendency  for  the  individual  to  contract  typhoid  fever,  and  on 
the  other,  if  the  disease  be  contracted,  the  likelihood  of  its 
having  a  fatal  result  is  diminished.  Thus  in  India,  of  4502 
soldiers  inoculated,  ,98  per  cent  contracted  typhoid,  while  of 
25,851  soldiers  in  the  same  stations  who  were  not  inoculated, 
2.54  per  cent  took  the  disease.  In  Ladysmith  during  the  siege 
there  were  1705  soldiers  inoculated,  among  whom  2  per  cent 
of  cases  occurred,  and  10,529  uninoculated,  among  whom 
14  per  cent  suffered  from  typhoid.  In  Harrismith,  Birt's 
statistics  show  that  in  typhoid  occurring  in  uninoculated  persons 
the  mortality  was  14.25  per  cent,  while  among  263  inoculated 
the  mortality  was  6.8  per  cent.  Wright  has  collected  statistics 
dealing  in  all  with  49,600  individuals,  of  whom  8600  were 
inoculated,  and  showed  a  case  incidence  of  2.25  per  cent,  with 
a  case  mortality  of  12  per  cent;  in  the  remaining  41,000  un- 
inoculated the  case  incidence  was  5.75  per  cent  and  the  case 
mortality  21  per  cent.  The  best  results  seem  to  be  obtained 
when  ten  days  after  the  first  inoculation  a  second  similar  inocu- 
lation is  practised.  Wright  has  found  that  in  certain  cases 
immediately  after  inoculation  there  is  a  fall  in  the  bactericidal 
power  of  the  blood,  and  he  is  of  opinion  that  this  indicates  a 
temporary  increased  susceptibility  to  the  disease.  He  therefore 
recommends  that  when  possible  the  vaccination  should  be 
carried  out  some  time  previous  to  the  exposure  to  infection. 
There  can  be  very  little  doubt  that  in  this  method  an  important 
prophylactic  measure  has  been  discovered. 

Anti-typhoid  Serum.  —  Bokenham  immunised  a  horse  by  filtered  bouillon 
cultures  of  the  typhoid  bacillus,  and  found  that  the  serum  had  neutralising 
power  for  the  bacilli  when  the  latter  mixed  with  it  were  injected  into  guinea- 
pigs.  When  injection  of  the  serum  was  followed  by  injection  of  bacilli,  the 
pathogenic  action  of  the  latter  was  to  a  certain  extent  prevented,  and  there 
was  also  evidence  of  the  serum  possessing  curative  properties. 

Methods  of  Examination.  —  The  methods  of  microscopic 
examination,  and  of  isolation  of  typhoid  bacilli  from  the  spleen 
post  mortem,  have  already  been  described.  They  may  be  iso- 


348  TYPHOID   FEVER. 

lated  from  the  Payer's  patches,  lymphatic  glands,  etc.,  by  a 
similar  method. 

During  life,  typhoid  bacilli  may  be  obtained  in  culture  in  the 
following  ways  :  — 

(a)  From  the  Blood.  —  As  stated  before,  several  observers 
have  shown  that  in  about  80  per  cent  of  all  cases  of  typhoid 
fever,  in  the  earlier  weeks  of  the  disease,  it  is  possible  to  obtain 
the  bacilli  from  the  blood  by  use  of  appropriate  methods  (see 
p.  72). 

(&)  From  the  Spleen. — This  is  the  most  certain  method  of 
obtaining  the  typhoid  bacillus  during  the  continuance  of  a  case. 
The  skin  over  the  spleen  is  purified,  and,  a  sterile  hypodermic 
syringe  being  plunged  into  the  organ,  there  is  withdrawn  from 
the  splenic  pulp  a  droplet  of  fluid,  from  which  plates  are  made. 
In  a  large  proportion  of  cases  of  typhoid  the  bacillus  may  be 
thus  obtained,  failure  only  occurring  when  the  needle  does  not 
happen  to  touch  a  bacillus.  Numerous  observations  have  shown 
that,  provided  the  needle  be  not  too  large,  the  procedure  is  quite 
safe.  Its  use,  however,  is  scarcely  called  for. 

(c)  From  the   Urine.  —  Typhoid  bacilli  are    present  in   the 
urine  in  about  twenty-five  per  cent  of  cases,  especially  late  in 
the  disease,   probably   chiefly  when  there    are   groups   in   the 
kidney  substance.     For  methods  of  examining  suspected  urine, 
see  p.  74. 

(d)  From  the  Stools.  —  During  the  first  ten  days  of  a  case  of 
typhoid  fever,  the  bacilli  can  be  isolated  from  the  stools  by  the 
ordinary   plate    methods  —  preferably    in    phenolated    gelatin. 
After  that  period,  though  the  continued  infectiveness  of  the 
disease  indicates  that  they  are  still  present,  their  isolation  is 
practically  hopeless. 

Numerous  special  media  have  from  time  to  time  been  devised  for  the  pur- 
pose of  readily  isolating  and  identifying  the  bacilli  from  the  stools.  The  most 
have  for  their  object  the  restraining  of  the  majority  of  intestinal  bacteria  by 
having  materials  incorporated  which  prove  unfavorable  to  their  development, 
whilst  readily  permitting  that  of  B.  typhosus.  Such  media  are  commonly 
known  by  their  author's  names,  e.g.,  Eisner,  Capaldi,  Remey,  Hiss,  Piorkow- 
ski,  Drigalski  and  Conradi  (see  references  in  chapter  on  Bibliography).  All 
are  more  or  less  of  doubtful  value  owing  to  difficulties  presented  in  acquiring 
proficiency  in  their  manufacture  or  application. 

We  have  seen  that  after  ulceration  is  fairly  established  by 
the  sloughing  of  the  necrosed  tissue,  the  numbers  present  in 


BACILLARY   DYSENTERY.  349 

the  patches  are  much  diminished  and  therefore  there  are  fewer 
cast  off  into  the  intestinal  lumen,  and  that  in  addition  there  is  a 
correspondingly  great  increase  of  the  B.  coli,  which  thus  causes 
any  typhoid  bacilli  in  a  plate  to  be  quite  outgrown.  From  the 
fact  that  the  ulcers  in  a  case  of  typhoid  may  be  very  few  in 
number,  it  is  evident  that  there  may  be  at  no  time  very  many 
typhoid  bacilli  in  the  intestine.  We  may  add  that  the  micro- 
scopic examination  of  the  stools  is  useless  as  a  means  of 
diagnosing  the  presence  of  the  typhoid  bacillus, 

Isolation  from  Water  Supplies.  —  A  great  deal  of  work  has 
been  done  on  this  subject.  It  is  evident  that  if  it  is  difficult  to 
isolate  the  bacilli  from  the  stools  it  must  a  fortiori  be  much  more 
difficult  to  do  so  when  the  latter  are  enormously  diluted  by 
water.  The  B.  typhosus  has,  however,  been  isolated  from  water 
during  epidemics.  This  was  done  by  Klein  in  the  outbreaks  in 
recent  years  at  Worthing  and  Rotherham.  The  B.  coli  is,  as 
might  be  expected,  the  organism  most  commonly  isolated  in 
such  circumstances.  In  the  case  of  both  bacteria,  the  whole 
series  of  culture  reactions  must  be  gone  through  before  any 
particular  organism  isolated  is  identified  as  the  one  or  the  other ; 
probably  there  are  saprophytes  existing  in  nature  which  only 
differ  from  them  in  one  or  two  reactions.  In  the  examination 
of  water,  the  addition  of  .2  per  cent  carbolic  acid  to  the  medium 
inhibits  to  a  certain  extent  the  growth  of  other  bacteria,  while 
the  B.  typhosus  and  the  B.  coli  are  unaffected.  In  examining 
waters,  the  ordinary  plate  methods  are  generally  used.  Klein, 
however,  niters  a  large  quantity  through  a  Berkefeld  filter,  and, 
brushing  off  the  bacteria  retained  on  the  porcelain,  makes 
cultures.  A  much  greater  concentration  of  the  bacteria  is  thus 
obtained.  On  the  whole  there  is  little  to  be  gained  from  this 
attempt  to  isolate  the  typhoid  bacillus  from  water  in  any 
particular  case,  and  it  is  much  more  useful  for  the  bacteriologist 
to  bend  his  energies  towards  the  obtaining  of  the  indirect  evi- 
dence of  contamination  of  water  by  sewage,  to  the  nature  of 
which  attention  has  been  called  in  Chapter  IV. 

BACILLARY  DYSENTERY. 

Dysentery  has  for  long  been  recognised  as  including  a  number 
of  different  pathological  conditions,  and  within  more  recent  times 


350  BACILLARY    DYSENTERY. 

amoebic  and  non-amoebic  forms  have  been  distinguished.  Of 
the  latter  bacteria  have  been  believed  to  be  the  causal  agents, 
and  an  organism  described  by  Shiga  in  1898  has  almost  been 
established  as  the  cause  of  a  large  proportion  of  cases.  Shiga's 
observations  were  made  in  Japan,  and  the  confirmatory  results 
obtained  by  Kruse  in  Germany,  by  Flexner  and  by  Strong  and 
Harvie  in  the  Philippine  Islands,  and  more  recently  by  Vedder 
and  Duval  in  the  United  States,  tend  to  show  that  the  distribu- 
tion of  the  specific  organism  is  world-wide.  The  last-mentioned 
observers  also  compared  the  bacteria  obtained  from  these  differ- 
ent localities  and  found  them  to  be  identical.  Further  interest 
is  attached  to  the  role  played  by  this  organism  by  the  researches 
of  Duval  and  Bassett,  who  seemingly  have  isolated  the  bacillus 
from  the  faeces  in  forty-two  cases  of  summer  diarrhoea  in  infants, 
and  from  scrapings  of  the  intestinal  mucosa  at  autopsy,  and  in  one 
case  from  the  mesenteric  glands  and  liver.  Spronck  working  in 
Holland  confirms  the  work  of  Duval  and  Bassett  by  similar  results 
in  three  cases  of  the  same  disease.  The  evidence  for  the  rela- 
tionship of  the  organism  to  the  disease  consists  chiefly  in  the 
apparently  constant  presence  of  the  organism  in  the  dejecta  in 
this  class  of  dysentery,  the  agglutination  of  this  organism  by  the 

serum  of  patients  suffering 

,•  from  the  disease,  and  in 

the  production  of  a  cura- 
tive serum  through  the 
immunising  of  sheep  and 
asses  with  pure  cultures  of 

I  the  bacillus.    The  relation 

of   amoebae   to  dysentery 
will  be  discussed  in  the 
X,  Appendix. 

Bacillus  dysenteriae 
(Shiga).  —  Morphological 
Characters. — This  bacillus 
morphologically  closely 

FIG.  n8A.  — B.  dysenteriae;    from  an  agar  cul-    resembles       the        typhoid 
ture  48   hours   old.      Stained   with   aniline-gentian-    KaHlliis      but      is     On      the 


violet,     x  looo. 

whole  somewhat  plumper, 

and    filamentous    forms    are    comparatively    rare   (Fig.    n8A). 
Involution  forms   sometimes  occur,  specially  in  glucose  agar. 


CULTURAL   CHARACTERS.  351 

Most  observers  have  found  no  trace  of  motility,  while  others  say 
that  it  is  slightly  motile.  Vedder  and  Duval  have,  however,  by 
modification  of  Van  Ermengem's  process,  demonstrated  the  pres- 
ence of  numerous  lateral  flagella,  which  are  of  great  fineness, 
but  of  considerable  length.  No  spore  formation  occurs ;  the 
organism  is  stained  readily  by  the  ordinary  dyes,  but  is  decolor- 
ised by  Gram's  method. 

Cultural  Characters.  —  In  these  also  considerable  resemblance 
is  presented  to  the  typhoid  bacillus.  In  gelatin  a  whitish  line  of 
growth  occurs  along  the  puncture,  but  the  superficial  film-like 
growth  is  usually  absent,  or  at  least  poorly  marked.  In  plate-cul- 
tures also  the  superficial  growths  are  smaller,  and  have  less  of  the 
film-like  character  than  those  of  the  typhoid  organism.  On  agar 
growth  occurs  as  a  smooth  film  with  regular  margins,  but  after 
two  or  three  days,  especially  if  the  surface  be  moist,  there  oc- 
curs, as  Vedder  and  Duval  have  described,  an  outgrowth  of 
lateral  offshoots  on  the  surface  of  the  medium.  In  agar  plates 
the  colonies  resemble  those  of  the  typhoid  organism,  being  of 
smaller  size  and  less  opaque  than  those  of  the  bacillus  coli. 
In  peptone  bouillon  a  uniform  haziness  is  produced  and  the 
indol  reaction  is  not  usually  given.  This  organism  does  not 
ferment  grape  and  other  sugars,  there  being  no  evolution  of  gas 
by  ordinary  methods.  In  litmus  'milk  there  is  developed  at  first 
a  slight  degree  of  acidity,  which  is  followed  by  a  phase  of  in- 
creased alkalinity  ;  no  coagulation  of  the  milk  >ccurs.  Qnpotato 
the  organism  forms  a  transparent  or  whitish  layer,  which,  how- 
ever, in  the  course  of  a  few  days  assumes  a  brownish-red  or 
dirty-gray  colour,  with  some  discoloration  of  the  potato  at  the 
margin  of  the  growth. 

Powers  of  Resistance.  —  The  bacillus  is  killed  by  ten  minutes' 
exposure  to  moist  heat  at  55°  C.  Shiga  gives  the  following  data 
regarding  the  action  of  some  chemical  solutions  upon  this  organ- 
ism :  a  5  per  cent  solution  of  carbolic  acid  destroys  it  in  a  few 
minutes,  whereas  a  I  per  cent  solution  requires  half  an  hour ;  a 
brief  exposure  to  a  ^TriolF  solution  of  bichloride  of  mercury  suf- 
fices to  kill  it ;  five  minutes'  exposure  in  a  10  per  cent  solution  of 
95  per  cent  alcohol  causes  its  destruction.  Exposure  to  direct 
sunlight  for  thirty  minutes  kills  the  bacillus.  Drying  is  resisted 
for  several  days. 

Relations  to  the  Disease.  —  This  organism  has  been  found  in 


352  BACILLARY   DYSENTERY. 

large  numbers  in  the  dejecta,  especially  in  acute  cases,  where 
it  may  be  present  in  almost  pure  cultures.  The  organism  does 
not  appear  to  spread  deeply  or  to  invade  the  general  circulation. 
In  the  more  chronic  cases  it  is  difficult  to  obtain  on  account  of 
the  large  number  of  the  bacillus  coli  and  other  bacteria  pres- 
ent. 

Pathology.  —  As  already  stated,  both  acute  and  chronic 
cases  are  marked  by  the  presence  of  this  organism.  In  the 
former,  where  death  may  occur  in  from  one  to  six  days,  the 
chief  changes,  according  to  Flexner,  are  a  marked  swelling  and 
corrugation  of  the  mucous  membrane,  with  haemorrhage  and 
pseudo-membrane  at  places.  There  is  extensive  coagulation- 
necrosis  with  nbrinous  exudation  and  abundance  of  polymorpho- 
nuclear  leucocytes,  and  the  structure  of  the  mucous  membrane, 
as  well  as  that  of  the  muscularis  mucosae,  is  often  lost  in  the 
exudation.  There  is  also  great  thickening  of  the  sub-mucosa, 
with  great  infiltration  of  leucocytes,  these  being  chiefly  of  the 
character  of  "  plasma  cells."  In  the  more  chronic  forms  the 
changes  correspond,  but  are  more  of  a  proliferative  character. 
The  mucous  membrane  is  granular,  and  superficial  areas  are 
devoid  of  epithelium,  whilst  ulceration  and  pseudo-membrane 
are  present  in  varying  degree. 

Agglutination.  —  All  the  above-mentioned  observers  agree 
regarding  the  agglutination  of  this  bacillus  by  the  serum  —  that 
is,  in  the  cases  of  dysentery  from  which  the  organism  can  be  cul- 
tivated. The  reaction  is  most  marked  after  from  six  to  seven 
days  in  the  acute  cases,  and  is  usually  given  in  a  dilution  of  from 
one  in  twenty  to  one  in  fifty  within  an  hour,  though  sometimes 
much  higher  dilutions  give  a  positive  result.  In  the  more 
chronic  cases  the  reaction  is  less  marked,  and  here  the  sedi- 
mentation method  is  to  be  preferred.  Agglutination  of  this 
organism  has  not  been  obtained  with  serum  from  cases  other 
than  those  of  dysentery,  nor  has  a  bacillus  been  cultivated  from 
other  such  sources.  The  reaction  is  also  absent  in  those  cases 
of  dysentery  which  are  manifestly  of  amoebic  nature  (p.  529). 

Pathogenic  Properties.  —  Mice  and  guinea-pigs  are  especially 
susceptible  both  to  subcutaneous  and  intraperitoneal  inoculation, 
dying  frequently  within  24  to  48  hours.  Cats  also  die  from 
subcutaneous  inoculation,  but  are  resistant  when  fed  with  the 
organism,  excepting  after  doses  of  croton  oil,  when  they  fre- 


METHOD    OF   ISOLATING   B.    DYSENTERIC.  353 

quently  succumb.  Rabbits  as  a  rule  recover  from  subcutaneous 
inoculation,  which  usually  produces  well-marked  local  swelling ; 
but  in  two  instances  Flexner  was  able  to  cause  death  in  rabbits 
following  upon  subcutaneous  inoculation,  both  with  a  Philippine 
culture  and  with  one  of  Kruse's,  and  in  each  animal  lesions  were 
found  in  the  colon  quite  analogous  to  those  seen  in  the  human 
subject.  Dogs  generally  die  within  5  to  6  days  after  being  fed 
with  cultures,  developing  well-marked  diarrhoea,  and  post  mortem 
the  large  intestine  is  usually  found  to  be  much  swollen.  Monkeys 
were  found  by  Flexner  to  be  most  resistant,  subcutaneous  inocu- 
lation producing  only  a  local  swelling,  which  rapidly  passed  away 
and  caused  no  apparent  illness,  even  large  doses  of  croton  oil 
followed  by  food  contaminated  with  the  bacilli  failed  to  produce 
infection. 

It  will  be  seen  that  the  evidence  furnished  is  practically  con- 
clusive as  to  the  causal  relationship  between  this  bacillus  and 
one  form  of  dysentery,  a  form,  moreover,  which  is  both  wide- 
spread and  embraces  a  large  proportion  of  cases  of  the  disease, 
and  especially  of  importance  is  the  fact  that  observations  made 
independently  in  different  countries  have  yielded  practically 
identical  results  on  this  point. 

Method  of  Examination.  —  So  far  as  is  known  the  bacilli  are 
found  only  in  the  dejecta,  especially  amongst  the  small  portions 
of  bloody  mucous  present  therein  in  acute  cases,  and  in  the  small 
shreds  of  mucous  membrane  should  these  be  found.  In  thirty- 
six  cases  examined,  Shiga  obtained  the  bacillus  in  thirty-four 
from  the  dejecta,  and  in  two  others  post  mortem  from  the 
intestinal  mucous  membrane.  Preferably  agar  plates  are  to  be 
employed  in  culture  work,  and  these  are  to  be  incubated  at 
37°  C.  Vedder  and  Duval  found  that  if  colonies  which  appeared 
after  twelve  hours  were  marked  with  a  pencil,  there  was  a 
greater  probability  of  obtaining  the  bacillus  of  dysentery  from 
those  which  appeared  later,  most  of  those  appearing  early  being 
colonies  of  bacillus  coli.  To  attain  an  early  recognition  of  the 
nature  of  these  later  appearing  colonies,  it  is  recommended  that 
sub-cultures  from  them  be  made  at  once  in  glucose  agar,  thereby 
differentiating  the  gas  formers  from  the  non-gas  formers  without 
loss  of  time.  It  is  desirable  in  conducting  an  examination  to 
obtain  sufficient  blood  of  the  patient  or  cadaver  to  enable  one  to 
carry  out  an  agglutination  test  upon  the  isolated  bacilli.  In  the 


354  BACILLARY   DYSENTERY. 

examination  of  chronic  cases  of  the  disease  post  mortem,  it  is 
usually  difficult  to  isolate  the  bacillus  on  account  of  the  large 
number  of  bacillus  coli  and  other  bacteria  present ;  in  such 
cases  it  is  advisable  to  scrape  the  ulcerated  mucosa  with  a 
sterile  knife  and  from  the  scrapings  make  numerous  dilutions 
in  agar.  Lactose  litmus  agar  may  be  found  helpful  in  differen- 
tiating colon  colonies  from  those  of  bacillus  dysenteriae  inasmuch 
as  the  former  colonies  appear  red  through  the  production  of 
lactic  acid,  whilst  the  latter,  not  forming  acid,  remain  blue. 

Bacillus  Dysenteriae  (Ogata).  —  Ogata  obtained  this  bacillus  in  an  ex- 
tensive epidemic  in  Japan  in  which  no  amoebae  were  present.  He  found  in 
sections  of  the  affected  tissues  enormous  numbers  of  small  bacilli  of  about  the 
same  thickness  as  the  tubercle  bacillus,  but  very  much  shorter.  These  bacilli 
were  sometimes  found  in  a  practically  pure  condition.  They  were  actively 
motile  and  could  be  stained  by  Gram's  method.  He  also  obtained  pure 
cultures  'from  various  cases  and  tested  their  pathogenic  effects.  They  grew 
well  on  gelatin  at  the  ordinary  temperature,  producing  liquefaction,  the  growth 
somewhat  resembling  that  of  the  cholera  spirillum.  By  injection  into  cats 
and  guinea-pigs,  as  well  as  by  feeding  them,  this  organism  was  found  to  have 
distinct  pathogenic  effects ;  these  were  chiefly  confined  to  the  large  intestine, 
haemorrhagic  inflammation  and  ulceration  being  produced.  It  still  remains  to 
be  determined  whether  this  organism  has  a  causal  relationship  to  one  variety 
of  dysentery. 

BACILLUS  ENTERITIDIS  SPOROGENES. 

This  organism  was  first  isolated  by  Klein  from  the  evacuations  in  an  out- 
break of  diarrhoea  following  the  ingestion  of  milk  which  contained  the 
microbe,  and  it  was  subsequently  found  by  him  in  certain  cases  of  infantile 
diarrhoea  and  of  summer  diarrhoea,  in  certain  instances  in  milk,  and  as  a 
constant  inhabitant  of  sewage  (see  Chapter  IV.).  In  films  made  from  the 
stools  in  diarrhoea  cases  where  it  is  present  it  can  be  microscopically  recog- 
nised as  a  bacillus  i.6/x  to  4.8/z  in  length  and  .8/x  in  breadth,  staining  by 
ordinary  stains  and  retaining  the  dye  in  Gram's  method.  It  often  contains  a 
spore  near  one  of  the  ends,  or  sometimes  nearer  the  centre.  It  is  slightly 
motile,  and  in  cultures  can  be  shown  to  possess  a  small  number  of  terminal 
flagella.  It  grows  well  under  anaerobic  conditions  on  ordinary  media,  espe- 
cially on  those  containing  reducing  agents.  On  agar  the  colonies  are  circular, 
grey,  and  translucent.-  and  under  a  low  power  are  seen  to  have  a  granular 
appearance.  On  this  medium  spore  formation  does  not  occur,  but  is  easily 
obtained  if  the  organism  is  grown  on  solidified  blood  serum,  which,  further,  is 
liquefied  during  growth.  On  gelatin  plates  liquefaction  commences  after 
twenty-four  hours  at  20°  C.  The  bacillus  grows  well  on  2  per  cent  dextrose 
gelatin,  and  besides  the  liquefaction  there  is  here  great  gas  evolution.  Spore 
formation  can  be  seen  to  take  place  in  this  medium,  but  the  degree  seems  to 
be  in  inverse  ratio  to  the  amount  of  gas  formation.  Very  typical  is  the  growth 


*"   OF 

UNIVERSITY 


B.  ENTERITIDIS    SPOROGENES^  355 

on  milk,  and  it  is  by  this  medium  that  isolation  can  be  best  effected.  A  small 
qijantity  of  the  material  suspected  to  contain  the  bacillus  is  placed  in  15  to 
20  c.c.  of  sterile  milk,  which  is  then  heated  for  ten  minutes  at  80°  C.  to  destroy 
all  vegetative  bacteria;  the  tube  is  cooled,  placed  under  anaerobic  conditions, 
and  incubated  at  37°  C.  for  from  twenty-four  to  thirty-six  hours.  If  the 
bacillus  be  present  there  is  abundant  gas  formation,  and  almost  complete 
separation  of  the  curd  from  the  whey  takes  place.  The  former  adheres  to  the 
sides  of  the  tube  in  shreds,  and  large  masses  gather  with  the  cream  on  the  top 
of  the  fluid,  all  being  torn  by  the  gas  evolved.  The  whey  is  only  slightly 
turbid  and  contains  numerous  bacilli.  The  growth  has  an  odour  of  butyric 
acid.  If  a  small  quantity  (say  i  c.c.)  of  the  whey  be  injected  into  a  guinea- 
pig,  the  animal  becomes  ill  in  a  few  hours  and  dies  in  twenty-four  hours.  At 
the  point  of  inoculation  the  skin  and  subcutaneous  tissues,  and  sometimes 
even  the  subjacent  muscles,  are  green  and  gangrenous  and  evil-smelling,  there 
is  considerable  oedema,  and  there  may  also  be  gas  formation.  The  exudation 
is  crowded  with  bacilli,  which,  however,  are  not  generally  distributed  in  any 
numbers  throughout  the  body.  These  pathogenic  properties  of  the  bacillus 
enteritidis  sporogenes  are  important  in  its  recognition,  for  its  culture  reactions 
taken  alone  are  very  similar  to  those  of  the  bacillus  butyricus  of  Botkin. 

Not  a  few  American  and  Continental  workers  exhibit  some  hesitancy  in 
accepting  the  status  of  B.  enteritidis  sporogenes  as  established  by  Klein;  for 
his  published  descriptions  are  not  free  from  the  suspicion  of  the  existence  of 
cultural  impurities  involved  in  the  technique  employed.  In  fact,  excepting 
the  presence  of  motility  and  flagella,  the  above  description  corresponds  closely 
to  that  of  B.  aerogenes  capsulatus  (Welch),  and  cultures  of  B.  enteritidis 
sporogenes  received  at  the  Pathological  Laboratory  of  the  Johns  Hopkins 
University,  through  the  courtesy  of  Dr.  Klein,  agreed  in  every  detail  to  pure 
cultures  of  B.  aerogenes  capsulatus  (Welch),  previously  described  by  Welch 
and  Nuttall. 


CHAPTER   XVI. 

DIPHTHERIA. 

THERE  is  no  better  example  of  the  valuable  contributions  of 
bacteriology  to  scientific  medicine  than  that  afforded  in  the  case 
of  diphtheria.  Not  only  has  research  supplied,  as  in  the  case  of 
tubercle,  a  means  of  distinguishing  true  diphtheria  from  condi- 
tions which  resemble  it,  but  the  study  of  the  toxins  of  the 
bacillus  has  explained  the  manner  by  which  the  pathological 
changes  and  characteristic  symptoms  of  the  disease  are  brought 
about,  and  has  led  to  the  discovery  of  the  most  efficient  means 
of  treatment,  namely,  the  anti-diphtheritic  serum. 

Historical.  —  As  in  the  case  of  many  other  diseases,  various  organisms 
which  have  no  causal  relation  to  the  disease  were  formerly  described  in  the 
false  membrane.  The  first  account  of  the  bacillus  now  known  to  be  the  cause 
of  diphtheria  was  given  by  Klebs  in  1883,  who  described  its  characters  in  the 
false  membrane,  but  made  no  cultivations.  It  was  first  cultivated  by  LofHer 
from  a  number  of  cases  of  diphtheria,  his  observations  being  published  in  1884, 
and  to  him  we  owe  the  first  account  of  its  characters  in  cultures  and  of  some 
of  its  pathogenic  effects  on  animals.  The  organism  is  for  these  reasons  known 
as  the  Klebs-Loffler  bacillus,  or  simply  as  Lofflers  bacillus.  By  experimental 
inoculation  with  the  cultures  obtained,  Loffler  was  able  to  produce  false  mem- 
brane on  damaged  mucous  surfaces,  but  he  hesitated  to  conclude  definitely 
that  this  organism  was  the  cause  of  the  disease,  for  he  did  not  find  it  in  all  the 
cases  of  diphtheria  examined,  he  was  not  able  to  produce  paralytic  phenomena 
in  animals  by  its  injection,  and,  further,  he  obtained  the  same  organism  from  the 
throat  of  a  healthy  child.  This  organism  became  the  subject  of  much  inquiry, 
but  its  relationship  to  the  disease  may  be  said  to  have  been  definitely  estab- 
lished by  the  brilliant  researches  of  Roux  and  Yersin,  who  made  an  extensive 
study  of  its  character  and  life  history,  and  showed  that  the  most  important 
features  of  the  disease  could  be  produced  by  means  of  the  separate  toxins  of  the 
organism.  Their  experiments  were  published  in  1888-90.  Further  light  has 
been  thrown  on  the  subject  by  the  work  of  Sidney  Martin,  who  has  found  that 
there  can  be  separated  from  the  organs  in  cases  of  diphtheria  substances  which 
act  as  nerve  poisons,  and  also  produce  other  phenomena  met  with  in 
diphtheria. 

General  Facts.  —  Without  giving  a  description  of  the  patho- 
logical changes  in  diphtheria,  it  will  be  well  to  mention  the 

356 


BACILLUS    DIPHTHERIA.  357 

outstanding  features  which  ought  to  be  considered  in  connection 
with  its  bacteriology.  In  addition  to  the  formation  of  false 
membrane,  which  may  prove  fatal  by  mechanical  effects,  the 
chief  clinical  phenomena  are  the  symptoms  of  general  poison- 
ing, great  muscular  weakness,  tendency  to  syncope,  and 
albuminuria;  also  the  striking  paralyses  which  occur  later  in 
the  disease,  and  which  may  affect  the  muscles  of  the  pharynx, 
larynx,  and  eye,  or  less  frequently  the  lower  limbs  (being  some- 
times of  paraplegic  type),  all  these  being  grouped  together 
under  the  term  "  post-diphtheritic  paralyses."  It  may  be  stated 
here  that  all  these  conditions  have  been  experimentally  repro- 
duced by  the  action  of  the  bacillus  of  diphtheria,  or  by  its 
toxins.  Other  bacteria  are,  however,  concerned  in  producing 
various  secondary  inflammatory  complications  in  the  region 
of  the  throat,  such  as  ulceration,  gangrenous  change,  and  sup- 
puration, which  may  be  accompanied  by  symptoms  of  general 
septic  poisoning. 

The  detection  of  the  bacillus  of  Loffler  in  the  false  mem- 
brane or  secretions  of  the  mouth  is  to  be  regarded  as  supplying 
the  only  certain  means  of  diagnosis  of  diphtheria.  With  the 
exception  of  the  tubercle  bacillus,  there  is  probably  no  organism 
which  has  been  the  subject  of  so  much  routine  examination,  and 
the  opinion  of  all  who  are  competent  to  judge  may  be  said  to  be 
unanimous  on  this  subject. 

Bacillus  Diphtherias.  —  Microscopical  Characters.  —  If  a  film 
preparation  be  made  from  a  piece  of  diphtheria  membrane  (in 
the  manner  described  below)  and  stained  with  methylene-blue, 
the  bacilli  are  found  to  have  the  following  characters.  They 
are  slender  rods,  straight  or  slightly  curved,  and  usually  about 
3  //,  in  length,  their  thickness  being  a  little  greater  than  that  of 
the  tubercle  bacillus.  The  size,  however,  varies  somewhat  in 
different  cases,  and  for  this  reason  varieties  have  been  distin- 
guished as  small  and  large,  and  even  of  intermediate  size.  It  is 
sufficient  to  mention  here  that  in  some  cases  most  are  about  3  //, 
in  length,  whilst  in  others  they  may  measure  fully  5  /*.  Corre- 
sponding differences  in  size  are  found  in  cultures.  They  stain 
deeply  with  the  blue,  sometimes  being  uniformly  coloured,  but 
often  showing,  in  their  substance,  little  granules  more  darkly 
stained,  so  that  a  dotted  or  beaded  appearance  is  presented. 
Sometimes  the  ends  are  swollen  and  more  darkly  stained  than 


358  DIPHTHERIA. 

the  rest;  often,  however,  they  are  rather  tapered  off  (Fig. 
119).  In  some  cases  the  terminal  swelling  is  very  marked,  so 
as  to  amount  to  clubbing,  and  with  some  specimens  of  methy- 

lene-blue  these 
swellings  and 
\  '  granules  stain 

t*          >*    1&  of  a  violet  tint. 

j»-  |     1  "  Distinct    club- 

Jt  **       A*          '   •       king,  however, 
*»  \  .         ^  f  *  is  less  frequent 


.  m 

v  P<^  ures.    There  is 


\  a  want  of  uni- 

1^^  "^    formity  in  the 

/4t      A  ,'  '  *  appearance  of 

the    bacilli    if 


<*  ~  j^''       compared  side 

by  side.  They 
usually  lie  ir- 
regularly scat- 
tered or  in 

FlG.  119.  —  Film  preparation  from  diphtheria  membrane  ;  show-  ' 

ing  numerous  diphtheria   bacilli.     One   or  two  degenerated  torrns  dividual  bacilli 

are  seen  near  the  centre  of  the  field.     (Cultures  made  from  the  same  ,      .          •> .  •, 

piece  of  membrane  showed  the  organism  to  be  present  in  practically  DCing  dispo 

pure  condition.)  in     all     direc- 

Stained  with  methylene-blue.     X  1000.  .  q 

may  be  contained  within  leucocytes.  They  do  not  form 
chains,  but  occasionally  forms  longer  than  those  mentioned  may 
be  found,  and  these  specially  occur  in  the  spaces  between  the 
fibrin  as  seen  in  sections. 

Distribution  of  the  Bacillus.  —  The  diphtheria  bacillus  may 
be  found  in  the  membrane  wherever  it  is  formed,  and  may  also 
occur  in  the  secretions  of  the  pharynx  and  larynx  in  the  disease. 
It  may  be  mentioned  that  distinctions  formerly  drawn  between 
true  diphtheria  and  non-diphtheritic  conditions  from  the  appear- 
ance and  site  of  the  membrane,  have  no  scientific  value,  the  only 
true  criterion  being  the  presence  of  the  diphtheria  bacillus. 
The  occurrence  of  a  membranous  formation  produced  by 
streptococci  has  already  been  t  mentioned  (p.  193). 

In  diphtheria  the  membrane  has  a  somewhat  different  struc- 


DISTRIBUTION   OF   THE   BACILLUS. 


359 


ture  according  as  it  is  formed  on  a  surface  covered  with  stratified 
squamous  epithelium  as  in  the  pharynx,  or  on  a  surface  covered 
by  ciliated  epithelium  as  in  the  trachea.  In  the  former  situa- 
tion necrosis  of  the  epithelium  occurs  either  uniformly  or  in 
patches,  and  along  with  this  there  is  marked  inflammatory  reac- 
tion in  the  connective  tissue  beneath,  attended  by  abundant 
fibrinous  exudation.  The  necrosed  epithelium  becomes  raised 
up  by  the  fibrin,  and  its  interstices  are  also  filled  by  it.  The 
fibrinous  exudation  also  occurs  around  the  vessels  in  the  tissue 


m  v 


FlG.  120. —  Section  through  a  diphtheritic  membrane  in  trachea,  showing  diphtheria 
bacilli  (stained  darkly)  in  clumps,  and  also  scattered  amongst  the  fibrin.  Some  strepto- 
cocci are  also  shown,  towards  the  surface  on  the  left  side. 

Stained  by  Gram's  method  and  Bismarck-brown.     X  1000. 

beneath,  and  in  this  way  the  membrane  is  firmly  adherent.  In 
the  trachea,  on  the  other  hand,  the  epithelial  cells  rapidly  become 
shed,  and  the  membrane  is  found  to  consist  almost  exclusively 
of  fibrin  with  leucocytes,  the  former  arranged  in  a  reticulated 
or  somewhat  laminated  manner,  and  varying  in  density  in  differ- 
ent parts.  The  membrane  lies  upon  the  basement  membrane, 
and  is  less  firmly  adherent  than  in  the  case  of  the  pharynx. 

The  position  of  the  diphtheria  bacilli  varies  somewhat  in 
different  cases,  but  they  are  most  frequently  found  lying  in  oval 
or  irregular  clumps  in  the  spaces  between  the  fibrin,  towards 


360  DIPHTHERIA. 

the  superficial,  that  is,  usually,  the  oldest  part  of  the  false  mem- 
brane (Fig.  120).  There  they  may  be  in  a  practically  pure 
condition,  though  streptococci  and  occasionally  some  other  or- 
ganisms may  be  present  along  with  them.  They  may  occur 
also  deeper,  but  are  rarely  found  in  the  fibrin  around  the  blood- 
vessels. On  the  surface  of  the  membrane  they  may  be  also 
seen  lying  in  large  numbers,  but  are  there  usually  accompanied 
by  numerous  other  organisms  of  various  kinds.  Occasionally 
a  few  bacilli  have  been  detected  in  the  lymphatic  glands.  As 
Loffier  first  described,  they  may  be  found  after  death  in  pneu- 
monic patches  in  the  lung,  this  being  a  secondary  extension  by 
the  air  passages.  They  have  also  been  occasionally  found  by 
various  observers  in  the  spleen,  liver,  and  other  organs  after 
death.  This  occurrence  is  probably  to  be  explained  by  an  en- 
trance into  the  blood  stream  shortly  before  death,  similar  to 
what  occurs  in  the  case  of  other  organisms,  e.g.  the  bacillus 
coli  communis.  The  diphtheria  bacillus  may  also  infect  other 
mucous  membranes.  It  is  found  in  true  diphtheria  of  the  con- 
junctiva, and  may  also  occur  in  similar  affections  of  the  vulva 
and  vagina :  some  of  these  cases  have  been  treated  success- 
fully with  diphtheria  antitoxin.  The  pseudo-diphtheria  bacillus, 
however,  may  also  occur  in  these  situations. 

Association  with  other  Organisms.  —  The  diphtheria  organ- 
ism is  sometimes  present  alone  in  the  membrane,  but  more  fre- 
quently is  associated  with  some  of  the  pyogenic  organisms,  the 
streptococcus  pyogenes  being  the  commonest.  The  staphylo- 
cocci,  and  occasionally  the  pneumococcus  or  the  bacillus  coli, 
may  be  present  in  some  cases.  Streptococci  are  often  found  ly- 
ing side  -by  side  with  the  diphtheria  bacilli  in  the.  membrane,  and 
also  penetrating  more  deeply  into  the  tissues.  In  some  cases  of 
tracheal  diphtheria  we  have  found  streptococci  alone  at  a  lower 
level  in  the  trachea  than  the  diphtheria  bacilli,  where  the  mem- 
brane was  thinner  and  softer,  the  appearance  in  these  cases  be- 
ing as  if  the  streptococci  acted  as  exciters  of  inflammation  and 
prepared  the  way  for  the  bacilli.  It  is  still  a  matter  of  dispute 
as  to  whether  the  association  of  the  diphtheria  bacillus  with  the 
pyogenic  organisms  is  a  favourable  sign  or  the  contrary,  though 
on  experimental  grounds  the  latter  is  the  more  probable.  We 
know,  however,  that  some  of  the  complications  of  diphtheria 
may  be  due  to  the  action  of  pyogenic  organisms.  The  exten- 


CULTIVATION   OF   B.    DIPHTHERIA. 


361 


sive  swelling  of  the  tissues  of  the  neck,  sometimes  attended  by 
suppuration  in  the  glands,  and  also  various  haemorrhagic  con- 
ditions, have  been  found  to  be  associated  with  their  presence ; 
in  fact,  in  some  cases  the  diphtheritic  lesion  enables  them  to  get 
a  foothold  in  the  tissues,  where  they  exert  their  usual  action  and 
may  lead  to  extensive  suppurative  change,  to  septic  poisoning,  or 
to  septicaemia.  In  cases  where  a  gangrenous  process  is  super- 
added,  a  great  variety  of  organisms  may  be  present,  some  of 
them  being  anaerobic. 

Against  such  complications  anti-diphtheritic  serum  produces 
no  favourable  effect,  as  its  action  is  specific  and  only  neutralises 
the  toxins  of  the  diphtheria  bacillus.  In  view  of  this  fact,  in 
some  cases  the  anti-streptococcic  serum  has  been  used  along  with 
it,  and  it  is  apparent  that  in  such  conditions  the  bacteriological 
examination  of  the  parts  affected  may  afford  valuable  indications 
as  to  treatment. 

Cultivation.  —  The  diphtheria  bacillus  grows  best  in  cultures 
at  the  temperature  of  the  body ;  growth  still  takes  place  at  22° 
C.,  but  ceases  at  20°  C.  The  best 
media  are  the  following  :  Lb'ffler's 
original  medium  (p.  45),  solidified 
blood  serum,  alkaline  blood  serum 
(Lorrain  Smith),  blood  agar,  and 
the  ordinary  agar  media.  If  in- 
oculations be  made  on  the  surface 
of  blood  serum  with  a  piece  of 
diphtheria  membrane,  colonies  of 
the  bacillus  appear  within  twenty- 
four  hours,  and  often  before  any 
other  growths  are  visible.  The 
colonies  are  small  circular  discs 
of  opaque,  whitish  colour,  their 
centre  being  thicker  and  of  darker 
greyish  appearance  when  viewed  by  transmitted  light  than  the 
periphery.  On  the  second  or  third  day  they  may  reach  3  mm. 
in  size,  but  when  numerous  they  remain  smaller.  Upon  agar 
plates  the  surface  colonies  at  the  end  of  twenty-four  to  forty-  >' 
eight  hours'  incubation  resemble  those  of  streptococcus  pyogenes, 
but  are  usually  larger  and  have  a  tendency  towards  wavy  mar- 
gins. Under  the  low  power  of  the  microscope  such  colonies 


a  b 

FIG.  121.  —  Cultures  of  the  diph- 
theria bacillus  on  an  agar  plate;  twenty- 
six  hours'  growth. 

(a)  Two  successive  strokes; 

(b)  isolated  colonies  from  the  same  plate. 


362  DIPHTHERIA. 

are  found  to  be  of   a  grey-yellow  colour,   translucent,   rather 
coarsely  granular,  often  nucleated  and  reticulated.     The  deep 


•** 


FIG.   122.  —  Diphtheria   bacilli    from    a  FlG.  123.  —  Diphtheria  bacilli  of  larger 

twenty-four  hours'  culture  on  agar.  size  than  in  previous  figure,  showing  also 

Stained  with  methylene-blue.     X  1000.          irregular  staining  of  protoplasm.     From  a 

three  days'  agar  culture. 

Stain  :  weak  carbol-fuchsin.     X  1000. 

colonies  show  nothing  very  striking.  In  stroke-cultures  the 
growth  forms  a  continuous  layer  of  the  same  dull  whitish  colour, 
the  margins  of  which  often  show  single  colonies  partly  or  com- 

pletely separated.  On  gelatin  at 
22°  C.  a  puncture  culture  shows 
a  line  of  dots  along  the  needle 
track,  whilst  at  the  surface  a 
small  disc  forms,  rather  thicker 
in  the  middle.  In  none  of  the 
media  does  any  liquefaction 
occur.  In  bouillon  the  organism 
produces  a  turbidity  which  soon 
settles  to  the  bottom  and  forms 
a  powdery  layer  on  the  wall 
of  the  vessel.  By  starting  the 
growth  on  the  surface  and 

rlG.  124.  —  Involution  forms  of  the  diph- 
theria  bacillus  ;  from  an  agar  culture  of  seven    keeping     the    flasks     at     rest     a 


carboMhionin-b.u,     x  ,000.  f°™S- 

especially  suitable  for  the  de- 

velopment  of  toxin.     Ordinary  bouillon   becomes  acid   during 
the  first  two  or  three  days,  and  several  days  later  again  acquires 


POWERS   OF   RESISTANCE.  363 

an  alkaline  reaction.  If,  however,  the  bouillon  is  glucose-free 
(p.  80)  the  acid  reaction  does  not  occur. 

In  these  media  the  bacilli  show  the  same  characters  as  in 
the  membrane,  but  the  irregularity  in  staining  is  more  marked 
(Figs.  122,  123).  They  are  at  first  fairly  uniform  in  size  and 
shape,  but  later  involution  forms  are  present.  Many  are 
swollen  at  their  ends  into  club-shaped  masses  which  stain 
deeply,  and  the  protoplasm  becomes  broken  up  into  globules 
with  unstained  parts  between  (Fig.  124).  Some  become  thicker 
throughout,  and  segmented  so  as  to  appear  like  large  cocci,  and 
others  show  globules  at  their  ends,  the  rest  of  the  rod  appearing 
as  a  faintly  stained  line.  Occasionally  branched  forms  are  met 
with  in  agar  or  blood-serum  cultures,  which  take  the  shape  of 
a  three-rayed  star  usually.  Lately,  Hill  has  demonstrated  the 
fact  that  even  more  complex  branching  forms  are  by  no  means 
rare  and  can  be  observed  to  occur  in  "  hanging-block  "  prepara- 
tions upon  a  warm  stage  within  the  course  of  a  few  hours. 
The  bacilli  are  non-motile,  and  do  not  form  spores. 

Staining.  —  They  take  up  the  basic  aniline  dyes,  e.g.  methy- 
lene-blue  in  watery  solution,  with  great  readiness,  and  stain 
deeply,  the  granules  often  giving  the  metachromatic  reaction 
as  described.  They  also  retain  the  colour  in  Gram's  method, 
though  they  are  more  easily  decolorised  than  the  pyogenic 
cocci. 

Neisser  has  recently  introduced  the  following  stain  as  an  aid  to  the  diagnosis 
of  the  diphtheria  bacillus.  Two  solutions  are  used  as  follows  :  (a}  i  grm. 
methylene-blue  (Grubler)  is  dissolved  in  20  c.c.  of  96  per  cent  alcohol,  and 
to  the  solution  are  added  950  c.c.  of  distilled  water  and  50  c.c.  of  glacial 
acetic  acid;  (b)  2  grms.  Bismarck-brown  (vesuvin)  dissolved  in  a  litre  of 
distilled  water.  Films  are  stained  in  (a)  for  1-3  seconds  or  a  little  longer, 
washed  in  water,  stained  for  3-5  seconds  in  (&),  dried,  and  mounted.  The 
protoplasm  of  the  diphtheria  bacillus  is  stained  a  faint  brown  colour.,  the 
granules  a  blue  colour.  Neisser  considers  that  this  reaction  is  characteristic 
*of  the  organism,  provided  that  cultures  on  Loffler's  serum  are  used  and  exam- 
ined after  9-24  hours'  incubation  at  34-35°  C,  but  more  extended  observations 
show  that  this  reaction  has  only  a  relative  value.  Satisfactory  results  are  not 
always  obtained  in  the  case  of  films  prepared  from  membrane,  etc.,  but  there 
is  no  doubt  that  here  also  the  method  is  one  of  considerable  value. 

Powers  of  Resistance,  etc.  —  In  cultures  the  bacilli  possess 
long  duration  of  life ;  at  the  room  temperature  they  may  survive 
for  two  months  or  longer.  In  the  moist  condition,  whether  in 


364  DIPHTHERIA. 

cultures  or  in  membrane,  they  have  a  low  power  of  resistance, 
being  killed  at  60°  C.  in  a  few  minutes.  On  the  other  hand,  in 
the  dry  condition,  they  have  great  powers  of  endurance.  In 
membrane  which  is  perfectly  dry,  for  example,  they  can  resist  a 
temperature  of  98°  C.  for  an  hour.  Dried  diphtheria  membrane, 
kept  in  the  absence  of  light  and  at  the  room  temperature,  has- 
been  proved  to  contain  diphtheria  bacilli  still  living  and  virulent 
at  the  end  of  several  months.  The  presence  of  light,  moisture, 
or  a  higher  temperature,  causes  them  to  die  out  more  rapidly. 
Corresponding  results  have  been  obtained  with  bacilli  obtained 
from  cultures  and  kept  on  dried  threads.  These  facts,  especially 
with  regard  to  drying,  are  of  great  importance,  as  they  show  that 
the  contagium  of  diphtheria  may  be  preserved  for  a  long  time 
in  the  dried  membrane. 

Effects  of  Inoculation.  —  In  considering  the  effects  produced 
in  animals  by  experimental  inoculations  of  pure  cultures,  we  have 
to  keep  in  view  the  local  changes  which  occur  in  diphtheria,  and 
also  the  symptoms  of  general  poisoning. 

Loffler  in  his  original  paper  stated  that  in  the  case  of  rabbits, 
guinea-pigs,  pigeons,  and  fowls,  the  bacilli  taken  from  pure 
cultures  produced  no  change  on  healthy  mucous  membranes,  but 
when  the  latter  were  injured  by  scarification  or  otherwise,  inocula- 
tion caused  the  formation  of  false  membrane.  A  similar  result 
was  obtained  when  the  trachea  was  inoculated  after  tracheotomy 
had  been  performed.  In  this  case  the  surrounding  tissues 
became  the  seat  of  a  blood-stained  oedema,  and  the  lymphatic 
glands  were  enlarged,  the  general  picture  resembling  pretty 
closely  that  of  laryngeal  diphtheria.  These  results  have  been 
amply  confirmed  by  other  observers.  The  membrane  produced 
by  such  experiments  is  usually  less  firm  than  in  human  diphtheria,, 
and  the  bacilli  are  not  generally  found  in  such  large  numbers  in 
the  membrane.  Rabbits  inoculated  after  tracheotomy  often  die, 
and  Roux  and  Yersin  were  the  first  to  observe  that  in  some 
cases  paralysis  may  appear  before  death. 

Subcutaneous  injection,  in  guinea-pigs,  of  diphtheria  bacilli 
in  a  suitable  dose,  produces  death  within  thirty-six  hours.  On 
section  at  the  site  of  inoculation  there  is  seen  a  small  patch  of 
greyish  membrane,  whilst  in  the  tissues  around  there  is  exten- 
sive inflammatory  oedema,  often  associated  with  haemorrhages, 
and  there  is  also  some  swelling  of  the  corresponding  lymphatic 


INOCULATION    EXPERIMENTS.  365 

glands.  The  internal  organs  show  general  congestion,  the 
supra-renal  capsules  being  especially  reddened  and  often  show- 
ing haemorrhage.  The  renal  epithelium  may  show  cloudy  swell- 
ing, and  there  is  often  effusion  into  the  pleural  cavities.  After 
injection  the  bacilli  increase  in  number  for  a  few  hours,  but 
multiplication  soon  ceases,  and  at  the  time  of  death  they  may  be 
less  numerous  than  when  injected.  The  bacilli  remain  practi- 
cally local,  cultures  made  from  the  blood  and  internal  organs 
giving  usually  negative  results,  though  sometimes  a  few  colonies 
may  be  obtained.  When  streptococci  or  staphylococci  are 
injected  at  the  same  time,  a  larger  number  of  diphtheria  bacilla 
enter  the  circulation  (Metin).  If  a  non-fatal  dose  of  a  culture 
be  injected,  a  local  necrosis  of  the  skin  and  subcutaneous  tissue 
may  follow  at  the  site  of  inoculation. 

In  rabbits,  after  subcutaneous  inoculation,  results  of  the  same 
nature  follow,  but  these  animals  are  less  susceptible  than  guinea- 
pigs,  and  the  dose  requires  to  be  proportionately  larger.  Roux 
and  Yersin  found  that  after  intravenous  injection  the  bacilli 
rapidly  disappeared  from  tin  blood,  and  even  after  the  injection 
of  i  c.c.  of  a  broth  culture  no  trace  of  the  organisms  could  be 
detected  by  culture  after  twenty-four  hours :  nevertheless  the 
animals  died  with  symptoms  of  general  toxaemia,  nephritis  also 
being  often  present  (cf.  "Cholera,"  p.  417).  The  dog  and  sheep 
are  also  susceptible  to  inoculation  with  virulent  bacilli,  but  the 
mouse  and  rat  enjoy  a  high  degree  of  immunity. 

Klein  found  that  cats  also  were  susceptible  to  inoculation.  The  animals 
usually  die  after  a  few  days,  and  post  mortem  there  is  well-marked  nephritis. 
He  also  found  that  after  subcutaneous  injection  in  cows,  a  vesicular  eruption 
appeared  on  the  teats  of  the  udder,  the  fluid  in  which  contained  diphtheria 
bacilli.  At  the  time  of  death  the  diphtheria  bacilli  were  still  alive  and  viru- 
lent at  the  site  of  injection.  The  most  striking  result  of  these  experiments  is 
that  the  diphtheria  bacilli  passed  into  the  circulation  and  were  present  in  the 
eruption  of  the  udder.  He  considers  that  this  may  throw  light  on  certain 
epidemics  of  diphtheria  in  which  the  contagion  was  apparently  carried  by  the 
milk.  Other  observers  have,  however,  failed  to  obtain  similar  results.  Dean 
and  Todd,  in  investigating  an  outbreak  of  diphtheria  traceable  to  milk  supplied, 
found  a  vesicular  eruption  on  the  teats  of  the  udder  in  which  diphtheria  bacilli 
were  present.  They,  however,  came  to  the  conclusion  that  these  bacilli  were 
not  the  cause  of  the  eruption,  but  were  the  result  of  a  secondary  contamination, 
probably  from  the  saliva  of  the  milkers.  The  occurrence  of  a  true  infection 
with  the  diphtheria  bacillus  in  the  horse  has  been  recently  described  by 
Cobbett. 


366  DIPHTHERIA. 

The  Toxins  of  Diphtheria.  —  As  in  the  above  experiments  the 
symptoms  of  poisoning  and  ultimately  a  fatal  result  occur  when 
the  bacilli  are  diminishing  in  number,  or  even  after  they  have 
practically  disappeared,  Roux  and  Yersin  inferred  that  the  chief 
effects  were  produced  by  toxins,  and  this  supposition  they 
proved  to  be  correct.  They  showed  that  broth  cultures  of  three 
or  four  weeks'  growth  freed  from  bacilli  by  filtration  were  highly 
toxic.  The  filtrate  when  injected  into  guinea-pigs  and  other 
animals  produces  practically  the  same  effects  as  the  living 
bacilli ;  locally  there  is  less  fibrinous  exudation  but  a  consider- 
able amount  of  inflammatory  oedema,  and,  if  the  animal  survive 
long  enough,  necrosis  in  varying  degree  of  the  superficial  tissues 
may  follow.  The  toxicity  may  be  so  great  that  .05  c.c.  or  even 
less  may  be  fatal  to  a  guinea-pig  in  twenty-four  hours. 

After  injection  either  of  the  toxin  or  of  the  living  bacilli, 
when  the  animals,  such  as  guinea-pigs,  rabbits,  dogs,  etc.,  survive 
long  enough,  paralytic  phenomena  may  occur.  The  hind  limbs 
are  usually  affected  first,  the  paralysis  afterwards  extending  to 
other  parts,  though  sometimes  the  fore  limbs  and  neck  first  show 
the  condition.  Sometimes  symptoms  of  paralysis  do  not  appear 
till  two  or  three  weeks  after  inoculation.  After  paralysis  has 
appeared,  a  fatal  result  usually  follows  in  the  smaller  animals, 
but  in  dogs  recovery  may  take  place.  There  is  evidence  that 
these  paralytic  phenomena  are  produced  by  toxoids,  i.e.  modified 
toxins  (p.  179),  as  they  may  occur  when  there  is  injected  along 
with  the  toxin  sufficient  antitoxin  to  neutralise  the  more  rapidly 
acting  toxin  proper.  One  point  of  much  interest  is  the  high 
degree  of  resistance  to  the  toxin  possessed  by  mice  and  rats. 
Roux  and  Yersin,  for  example,  found  that  2  c.c.  of  toxin,  which 
was  sufficient  to  kill  a  rabbit  in  sixty  hours,  had  no  effect  on  a 
mouse,  whilst  of  this  toxin  even  ^  c.c.  produced  extensive 
necrosis  of  the  skin  of  the  guinea-pig. 

Preparation  of  the  Toxin.  —  The  obtaining  of  a  very  active 
toxin  in  large  quantities  is  an  essential  in  the  preparation  of 
anti-diphtheritic  serum.  Certain  conditions  favour  the  develop- 
ment of  a  high  degree  of  toxicity,  viz.,  a  free  supply  of  oxygen, 
the  presence  of  a  large  proportion  of  peptone  or  albumin  in  the 
medium,  and  the  absence  of  substances  which  produce  an  acid 
reaction.  In  the  earlier  work  a  current  of  sterile  air  was  made 
to  pass  over  the  surface  of  the  medium,  as  it  was  found  that  by 


NATURE   OF   THE   TOXIN.  367 

this  means  the  period  of  acid  reaction  was  shortened  and  the 
toxin  formation  favoured.  This  expedient  is  now  considered 
unnecessary  if  an  alkaline  medium  free  from  glucose  is  used,  as 
in  this  no  acid  reaction  is  developed  :  it  is  then  sufficient  to  grow 
the  cultures  in  shallow  flasks.  The  absence  of  glucose  —  an  all- 
important  point  —  may  be  attained  by  the  method  described 
above  (p.  80),  or  by  using  for  the  preparation  of  the  meat  extract 
flesh  which  is  just  commencing  to  putrefy  (Spronck).  L.  Mar- 
tin uses  a  medium  composed  of  equal  parts  of  freshly  pre- 
pared peptone  (by  digesting  pigs'  stomachs  with  HC1  at  35° 
C),  and  glucose-free  veal  bouillon.  In  this  medium  he  has 
obtained  a  toxin  of  which  -g-J-g-  c.c.  is  the  fatal  dose  to  a  guinea- 
pig  of  500  grms.  He  finds  that  glucose,  glycerin,  saccharose, 
and  galactose  lead  to  the  production  of  an  acid  reaction,  whilst 
glycogen  does  not.  The  latter  fact  explains  how  some  ob- 
servers have  found  that  bouillon  prepared  from  qitite  fresh  flesh 
is  suitable  for  toxin  formaticjn.  There  is  in  all  cases  a  period  at 
which  the  toxicity  reaches  a  maximum,  usually  in  2-3  weeks, 
occurring  earlier  the  more  rabidly  the  toxin  is  formed ;  later  the 
toxicity  diminishes.  Martin  found  that  in  his  medium  the  maxi- 
mum was  reached  on  the  8th-ioth  day.  It  may  be  added  that 
the  power  of  toxin  formation  varies  much  in  different  races  of 
the  diphtheria  bacillus,  and  that  many  may  require  to  be  tested 
ere  one  suitable  is  obtained. 

Properties  and  Nature  of  the  Toxin.  —  The  toxic  substance 
in  filtered  cultures  is  a  relatively  unstable  body.  When  kept  in 
sealed  tubes  in  the  absence  of  light,  it  may  preserve  its  powers 
little  altered  for  several  months,  but,  on  the  other  hand,  it  grad- 
ually loses  them  when  exposed  to  the  action  of  light  and  air. 
Heating  at  58°  C.  for  two  hours  destroys  the  toxic  properties  in 
great  part,  but  not  altogether.  When,  however,  the  toxin  is 
evaporated  to  dryness,  it  has  much  greater  resistance  to  heat. 
One  striking  fact,  discovered  by  Roux  and  Yersin,  is  that  after 
an  organic  acid,  such  as'tartaric  acid,  is  added  to  the  toxin  the 
toxic  property  disappears,  but  that  it  can  be  in  great  part  re- 
stored by  again  making  the  fluid  alkaline. 

The  toxic  body  in  filtered  cultures  can  be  precipitated  by 
alcohol,  and  is  also  carried  down  by  calcium  phosphate.  It  is, 
however,  soluble  in  water  and  dialyses  somewhat  slowly  through 
animal  membranes.  By  repeated  precipitation  and  again  dis- 


368  DIPHTHERIA. 

solving,  aided  by  dialysis,  a  solution  is  obtained  which,  on 
evaporating  to  dryness,  gives  a  whitish-yellow  powder  contain- 
ing the  toxic  body,  though  not  in  a  chemically  pure  condition. 
From  the  characters  described,  Roux  and  Yersin  considered  that 
it  belonged  to  the  group  of  diastases  or  enzymes. 

The  true  chemical  nature  of  the  diphtheria  toxin  is  still  un- 
known, and  the  matter  is  further  complicated  by  the  possibility 
that  if  a  ferment  is  formed  by  the  bacilli  it  may  produce  other 
toxic  bodies  of  a  non-diastatic  nature.  Guinochet  showed  that 
toxin  was  also  formed  by  the  bacilli  when  grown  in  urine  with 
no  proteid  bodies  present.  After  growth  had  taken  place  he 
could  not  detect  proteid  bodies  in  the  fluid,  but  on  account  of 
the  very  minute  amount  of  toxin  present,  their  absence  could 
not  be  excluded.  Uschinsky  also  found  that  toxic  bodies  were 
produced  by  diphtheria  bacilli  when  grown  in  a  proteid-free 
medium.1  It  follows  from  this  that  if  the  true  toxin  is  a  proteid, 
it  may  be  formed  by  synthesis  within  the  bodies  of  the  bacilli,  as 
well  as  by  a  change  in  the  proteids  of  the  culture  fluid.  Brieger 
and  Boer  have  separated  from  diphtheria  cultures  a  toxic  body 
which  gives  no  proteid  reaction  (vide  p.  174). 

Toxic  bodies  have  also  been  obtained  from  the  tissues  of 
those  who  have  died  from  diphtheria.  Roux  and  Yersin,  by  using 
a  filtered  watery  extract  from  the  spleen  from  very  virulent  cases 
of  diphtheria,  produced  in  animals  death  after  wasting  and 
paralysis,  and  also  obtained  similar  results  by  employing  the 
urine.  The  subject  of  toxic  bodies  in  the  tissues  has,  however, 
been  specially  worked  out  by  Sidney  Martin.  He  has  separated 
from  the  tissues,  and  especially  from  the  spleen,  of  patients  who 
have  died  from  diphtheria,  by  precipitation  with  alcohol,  chemical 
substances  of  two  kinds,  namely,  albumoses  (proto-  and  deutero-, 
but  especially  the  latter),  and  an  organic  acid.  The  albumoses, 
when  injected  into  rabbits,  especially  in  repeated  doses,  produce 
fever,  diarrhoea,  paresis,  and  loss  of  weight,  with  ultimately  a 
fatal  result.  As  in  the  experiments  with  the  toxin  from  cultures, 
the  posterior  limbs  are  first  affected  ;  afterwards  the  respiratory 
muscles,  and  finally  the  heart,  are  implicated.  He  further  found 

1  Uschinsky's  medium  has  the  following  composition:  water,  1000  parts; 
glycerin,  30-40;  sodium  chloride,  5-7;  calcium  chloride,  .1;  magnesium  sulphate, 
.2— .4;  di-potassium  phosphate,  .2— .25;  ammonium  lactate,  6—7;  sodium  asparagi- 
nate,  3-4. 


VARIATIONS   IN   VIRULENCE   OF   THE    BACILLUS.       369 

that  this  paresis  is  due  to  well-marked  changes  in  the  nerves. 
The  medullary  sheaths  first  become  affected,  breaking  up  into 
globules ;  ultimately  the  axis  cylinders  are  involved,  and  may 
break  across,  so  that  degeneration  occurs  in  the  peripheral 
portion  of  the  nerve  fibres.  Such  changes  occur  irregularly  in 
patches,  both  sensory  and  motor  fibres  being  affected.  Fatty 
change  takes  place  in  the  associated  muscle  fibres.  There  may 
also  be  a  similar  condition  in  the  cardiac  muscle.  The  organic 
acid  has  a  similar  but  weaker  action.  Substances  obtained  from 
diphtheria  membrane  have  an  action  like  that  of  the  bodies 
obtained  from  the  spleen,  but  in  higher  degree.  Martin  con- 
siders that  this  is  due  to  the  presence  in  the  membrane  of  an 
enzyme  which  has  a  proteolytic  action  within  the  body,  resulting 
in  the  formation  of  poisonous  albumoses.  According  to  this 
view  the  actual  -toxic  bodies  are  not  the  direct  product  of  the 
bacillus,  but  are  formed  by  the  enzyme  which  is  produced  by  it 
locally  in  the  membrane.  Cartwright  Wood  has  also  found  that 
when  diphtheria  cultures  in  an  albumin-containing  medium  are 
filtered  germ-free  and  exposed  to  65°  C.  for  an  hour  (the  supposed 
ferments  being  thus  destroyed),  there  still  remain  albumoses 
which  produce  febrile  reaction  and  are  active  in  developing 
immunity.  The  existence  of  ferments,  though  a  possibility, 
cannot,  however,  be  considered  to  be  yet  completely  proved. 
Nor  is  it  yet  certain  whether  the  proteids  obtained  by  precipita- 
tion from  cultures  and  from  the  tissues  are  in  themselves  toxic, 
or  whether  the  toxic  bodies  are  carried  down  along  with  them. 

Immunity.  —  This  is  described  in  the  general  chapter  on 
Immunity.  It  is  sufficient  to  state  here  that  a  high  degree  of 
immunity,  against  both  the  bacilli  and  their  toxins,  can  be  pro- 
duced in  various  animals  by  gradually  increasing  doses  either 
of  the  bacilli  or  of  their  filtered  toxins  (vide  Chapter  XX.). 

Variations  in  the  Virulence  of  the  Diphtheria  Bacillus.  —  In 
cultures  on  serum  the  diphtheria  bacilli  retain  their  virulence 
fairly  well,  but  they  lose  it  much  more  quickly  on  less  suitable 
media,  such  as  glycerin  agar.  Roux  and  Yersin  found  that, 
when  the  bacilli  were  grown  at  an  abnormally  high  temperature, 
namely,  39.5°  C.,  and  in  a  current  of  air,  the  virulence  diminished 
so  much  that  they  became  practically  innocuous.  When  the 
virulence  was  much  diminished,  these  observers  found  that  it 
could  be  restored  if  the  bacilli  were  inoculated  into  animals  along 

2B 


3/0  DIPHTHERIA. 

with  streptococci,  inoculation  of  the  bacilli  alone  not  being  suc- 
cessful for  this  purpose.  If,  however,  the  virulence  had  fallen 
very  low,  even  the  presence  of  the  streptococci  was  insufficient 
to  restore  it.  As  a  rule,  the  cultures  most  virulent  to  guinea- 
pigs  are  obtained  from  the  gravest  cases  of  diphtheria,  though 
to  this  rule  there  are  frequent  exceptions.  It  has  been  abun- 
dantly established  that  after  the  cure  of  the  disease,  the  bacilli 
may  persist  in  the  mouth  for  weeks  and  months,  though  they 
often  quickly  disappear.  Roux  and  Yersin  found,  by  making 
cultures  at  various  stages  after  the  termination  of  the  disease, 
that  these  bacilli  in  the  mouth  gradually  become  attenuated. 
These  observations  are  of  importance  in  relation  to  the  subject 
of  the  pseudo-diphtheria  bacillus.  At  present  it  would  scarcely 
be  safe  to  make  a  definite  statement  as  regards  the  relation  of 
virulence  to  the  size  of  the  bacilli.  Perhaps  the  majority  of 
observers  have  found  that  the  bacilli  of  the  larger  form  are 
usually  more  virulent  than  those  of  the  shorter  form ;  but  this 
is  not  invariably  the  case,  as  sometimes  short  forms  are  obtained 
which  possess  an  extremely  virulent  character.  Both  the  long 
and  the  short  forms  may  become  attenuated  in  the  same  way. 

The  so-called  Pseudo-diphtheria  Bacillus.  —  Under  this  term 
more  than  one  species  of  bacillus  has  been  described  and  con- 
siderable confusion  has  arisen,  (a)  The  name  has  been  applied 
by  some  observers  to  an  organism  differing  from  the  diphtheria 
bacillus  solely  in  its  want  of  virulence.  Such  an  organism  must 
be  regarded  merely  as  the  diphtheria  bacillus  in  an  attenuated 
condition,  and  should  be  spoken  of  as  such,  (b}  On  the  other 
hand,  there  have  been  cultivated  several  species  of  bacilli  which 
resemble  the  diphtheria  bacillus  in  some  respects,  but  differ  from 
it  in  certain  important  points.  They  have  not  all  the  morpho- 
logical and  staining  characters  in  young  cultures,  and  do  not 
produce  an  acid  reaction  in  broth  containing  glucose  ;  along 
with  these  characters  minor  differences  in  cultures  may  be 
present.  Such  organisms  have  been  cultivated  from  the  throat, 
both  in  the  healthy  condition,  in  non-diphtheritic  affections, 
and  also  in  true  diphtheria  along  with  the  diphtheria  bacillus. 
The  term  "  pseudo-diphtheria  "  if  used  at  all  should  be  applied 
to  such  organisms.  The  type  most  commonly  met  with  is  a 
shorter  bacillus  than  the  diphtheria  bacillus,  with  usually  a 
single  unstained  septum  running  across  it,  though  sometimes 


PSEUDO-DIPHTHERIA   BACILLUS.  371 

there  may  be  more  than  one  (Fig.  125);  it  does  not  form  acid 
from  glucose,  is  non-pathogenic 
to  the  guinea-pig,  and  its  col- 
onies after  a  time  tend  to  become 
whiter  and  more  opaque  than 
those  of  the  diphtheria  bacillus. 
Involution  forms  may  some- 
times be  produced  by  it.  The 
name  "  Hof  mann's  bacillus  "  is 
often  used  to  denote  such  an 
organism.  This  organism  is  of 
comparatively  common  occur- 
rence:  Cobbett  found  it  157 
times  in  an  examination  of  692 

FIG.    125.  —  Pseudo-diphtheria     bacillus 
persons,  Ot  Whom  650  Were  not    (Hofmann's).     Young  agar  culture. 

suffering  from  diphtheria.  Stained  with  thionin-biue.    x  1000. 

Loffler,  in  1887,  was  the^  first  to  describe  a  bacillus  having  closely  the 
characters  of  the  diphtheria  bacillus,  but  differing  from  it  in  its  want  of 
virulence.  He  looked  upon  it  as  a  distinct  species,  and  gave  it  the  name 
of the  pseudo-diphtheria  bacillus.  Hofmann,  in  1888,  published  an  account  of 
his  investigations  on  this  subject.  He  obtained  the  pseudo-diphtheria  bacillus 
from  the  throat  in  healthy  conditions,  and  also  in  non-diphtheritic  affections. 
His  conclusions  with  regard  to  the  distinct  character  of  this  bacillus  were 
similar  to  those  of  LofBer.  Since  that  time  the  organism  has  been  the  subject 
of  much  research  and  discussion.  Roux  and  Yersin,  on  the  other  side,  found 
a  "pseudo-diphtheria"  bacillus  corresponding  in  all  its  characters  with  a 
greatly  attenuated  diphtheria  bacillus,  and  concluded  that  it  was  really  of  the 
same  nature.  They  failed  to  make  it  virulent  by  any  method ;  but  this  result 
was  also  obtained  in  the  case  of  artificially  attenuated  diphtheria  bacilli. 
Biggs  has  found  that  there  are  two  varieties  of  pseudo-diphtheria  bacilli,  both 
differing  from  the  true  diphtheria  bacillus ;  one  of  these  produces  an  acid 
reaction  in  broth  containing  glucose,  whilst  the  other  does  not.  According 
to  his  statistics  the  two  varieties  appear  to  occur  with  about  the  same  frequency, 
and  these  observations  have  been  in  the  main  confirmed  by  Cobbett  and 
Phillips.  Hewlett  and  Knight  find  evidence  that  a  true  diphtheria  bacillus 
may  be  modified  so  as  to  show  the  microscopic  and  cultural  characters  of  the 
pseudo-diphtheria  type,  this  evidence  being  obtained  both  by  successive  exam- 
inations of  the  throat  after  diphtheria  and  by  modifying  cultures  artificially. 
They  also  claimed  to  have  in  one  instance  transformed  a  bacillus  of  the 
Hofmann  type  into  a  genuine  diphtheria  bacillus.  Richmond  and  Salter,  by 
the  passage  through  finches  and  other  birds,  also  record  successful  transforma- 
tion of  a  Hofmann  bacillus  into  the  true  B.  diphtheriae.  Lesieur,  in  a  recent 
and  exhaustive  study  upon  the  relationship  existing  between  B.  diphtheriae 
and  the  so-called  B.  pseudo-diphthericus,  records  several  interesting  facts. 


3/2  DIPHTHERIA. 

By  the  long-continued  action  of  diffuse  daylight  in  a  dry  room,  he  was  enabled 
to  transform  three  species  of  virulent  B.  diphtherias  into  forms  which  were 
non-virulent  and  which  could  not  be  distinguished  from  the  real  Hofmann 
bacillus.  And  again,  by  the  aid  of  collodion  sac  cultures  passed  through  one 
or  two  generations  in  the  peritoneal  cavities  of  rabbits  ;  by  repeatedly  trans- 
ferring cultures  of  B.  pseudo-diphthericus  every  second  or  third  day  to 
nutrient  broth  and  incubating  at  37°  C. ;  and  by  cultivating  the  bacilli  in  broth 
with  staphylococcus  pyogenes  aurens,  he  was  able  to  transform  these  apparent 
Hofmann  bacilli  into  bacilli  having  the  characters  of  B.  diphtherias,  proving 
virulent  to  guinea-pigs,  and  having  their  virulence  counteracted  by  antitoxin. 
Some  pseudo-diphtheria  bacilli,  however,  resisted  all  attempts  at  alteration, 
and  Lesieur  therefore  concludes  that  the  majority  of  non-virulent  diphtheria- 
like  bacilli  met  with  are  probably  true  B.  diphtherias,  which,  through  unknown 
conditions,  have  lost  virulence  and  undergone  morphological  alterations  ;  the 
minority,  only,  can  lay  claim  to  the  term  "  pseudo-diphtheria  "  bacilli,  and 
probably  include  several  species. 

As  a  rule  the  appearances  of  the  colonies  and  the  micro- 
scopical characters  enable  a  rapid  diagnosis  to  be  made  in 
suspected  diphtheria  cases.  In  some  cases,  however,  difficulty 
may  be  met  with ;  and  in  the  first  place,  all  the  minor  cultural 
characters  must  be  carefully  examined,  including  the  reaction 
produced  in  broth.  By  this  procedure  it  may  be  determined 
whether  the  organism  in  question  differs  in  any  points  from  the 
diphtheria  bacillus.  A  positive  result  on  inoculating  a  guinea- 
pig  (say  with  I  c.c.  of  a  24  hours'  broth  culture)  will  be  con- 
clusive, but  we  consider  that  for  all  practical  purposes  an 
organism  having  all  the  microscopical  and  cultural  characters  of 
the  diphtheria  bacillus  may  be  accepted  as  such.  Even  if  it  is 
non-virulent,  it  is  probably  only  an  attenuated  diphtheria  bacillus. 
L.  Martin,  moreover,  has  recently  pointed  out  that  some  races 
of  diphtheria  bacillus  are  so  attenuated  that  I  c.c.  of  a  24  hours' 
growth  in  bouillon  does  not  cause"  death  in  a  guinea-pig,  yet  the 
true  nature  is  shown  not  only  by  their  microscopical  characters, 
etc.,  but  also  by  the  fact  that  on  more  prolonged  growth  they 
form  small  quantities  of  toxin,  which  is  neutralised  by  diphtheria 
antitoxin.  Neisser  also,  as  the  result  of  an  extended  inquiry, 
comes  to  a  similar  conclusion  with  regard  to  the  virulence,  and 
considers  that  the  characteristic  staining,  the  morphological 
characters,  and  the  production  of  acid  in  glucose  broth,  when 
taken  together,  afford  conclusive  evidence  as  to  the  identity  of 
the  diphtheria  bacillus. 

The  question,  however,  has  a  special  interest  in  regard  to  the 


SUMMARY    OF   PATHOGENIC   ACTION.  373 

origin  and  spread  of  the  disease.  As  is  well  known,  the  disease 
usually  spreads  by  infection,  direct  or  indirect,  from  patient  to 
patient ;  but  sometimes  it  appears  to  start  afresh,  as  it  were. 
In  the  latter  case  the  existence  of  the  non-virulent  diphtheria 
bacilli  may  possibly  afford  an  explanation  of  the  occurrence,  as. 
such  bacilli  are  sometimes  found  even  in  healthy  subjects.. 
The  possibility  of  the  transformation  of  the  pseudo-diphtheria. 
("  Hofmann's  " )  into  the  true  diphtheria  bacillus  has  been  the 
subject  of  much  controversy,  but  it  cannot  be  regarded  as 
sufficiently  established  that  such  a  transformation  may  be 
effected,  still  less  that  the  former  organism  is  related  to  the 
origin  and  spread  of  diphtheria. 

Xerosis  bacillus.  —  The  term  has  been  given  to  an  organism  first  observed  by 
Kuschbert  and  Neisser  in  xerosis  of  the  conjunctiva,  and  which  has  been  since 
found  in  many  other  affections  of  the  conjunctiva  and  even  in  normal  condi- 
tions. Morphologically  it  is  practically  similar  to  the  diphtheria  bacillus,  and 
even  in  cultures  presents  very  minor  differences.  It  is,  however,  non-virulent  to 
animals,  and,  according  to  Eyre,  does  not  produce  an  acid  reaction  in  neutral 
bouillon ;  in  this  way  it  can  be  distinguished  from  the  diphtheria  bacillus. 

Action  of  the  Diphtheria  Bacillus. — Summary. —  From  a 
study  of  the  morbid:  changes  in  diphtheria  and  of  the  results 
produced  experimentally  by  the  bacillus  and  its  toxins,  the 
following  summary  may  be  given  of  its  action  in  the  body. 
Locally,  the  bacillus  produces  inflammatory  change  with  fibrin- 
ous  exudation,  but  at  the  same  time  cellular  necrosis  is  also  an 
outstanding  feature.  Though  false  membranes  have  not  been 
produced  by  the  toxins,  a  necrotic  action  may  result  when  these 
are  injected  subcutaneously.  The  toxins  also  act  upon  the 
blood-vessels,  and  hence  oedema  and  tendency  to  haemorrhage 
are  produced ;  this  action  on.  the  vessels  is  also  exemplified  by 
the  general  congestion  of  organs.  The  hyaline  change  in  the 
walls  of  arterioles  and  capillaries  so  often  met  with  in  diphtheria 
is  another  example  of  the  action  of  the  toxin.  The  toxins  have 
also  a  pernicious  action  on  highly  developed  cells  and  on  nerve 
fibres.  Thus,  in  the  kidney,  cloudy  swelling  occurs,  which  may 
be  followed  by  actual  necrosis  of  the  secreting  cells,  and  along 
with  these  changes  albuminuria  is  present.  The  action  is  also 
well  seen  in  the  case  of  the  muscle  fibres  of  the  heart,  which 
may  undergo  a  sort  of  hyaline  change,  followed  by  granular  dis- 
integration or  by  an  actual  fatty  degeneration.  These  changes 


374  DIPHTHERIA. 

are  of  great  importance  in  relation  to  heart  failure  in  the  disease. 
Changes  of  a  somewhat  similar  nature  have  been  recently 
observed  in  the  nerve  cells  of  the  central  nervous  system,  those 
lying  near  the  capillaries,  it  is  said,  being  affected  first.  There 
is  also  the  striking  change  on  the  peripheral  nerves,  which  is 
shown  first  by  the  disintegration  of  the  medullary  sheaths,  as 
already  described.  It  is,  however,  still  a  matter  of  dispute  to 
what  extent  these  nerve  lesions  are  of  primary  nature  or 
secondary  to  changes  in  the  nerve  cells. 

Methods  of  Diagnosis. —  The  bacteriological  diagnosis  of 
diphtheria  depends  on  the  discovery  of  the  bacillus.  As  the 
bacillus  occurs  in  largest  numbers  in  the  membrane,  a  portion  of 
this  should  be  obtained  whenever  it  is  possible,  and  transferred 
to  a  sterile  test-tube.  (The  tube  can  be  readily  sterilised  by 
boiling  some  water  in  it.)  If,  however,  membrane  cannot  be 
obtained,  a  scraping  of  the  surface  with  a  platinum  loop  may  be 
sufficient.  Where  the  membrane  is  confined  to  the  trachea  the 
bacilli  are  often  present  in  the  secretions  of  the  pharynx,  and 
may  be  obtained  from  that  situation  by  swabbing  it  with  cotton 
wool  (non-antiseptic),  the  swab  being  put  into  a  sterile  tube  or 
bottle  for  transport.  A  convenient  method,  is  to  twist  a  piece 
of  cotton  wool  round  the  roughened  end  of  a  piece  of  very  stout 
iron  wire/ six  inches  long,  and  pass  the  other  end  of  the  latter 
through  a  cotton  plug  inserted  in  the  mouth  of  a  test-tube 
(compare  Fig.  54,  the  wire  taking  the  place  of  the  pipette),  and 
sterilise.  In  use  the  wire  and  plug  are  extracted  in  one  piece, 
and  after  swabbing  are  replaced  in  the  tube  for  transit.  A 
scraping  may  be  made  off  the  swab  for  microscopic  examination, 
and  the  swab  may  be  smeared  over  the  surface  of  a  serum  tube 
to  obtain  a  culture. 

The  means  for  identifying  the  bacillus  are  (a)  By  microscopical 
examination. —  For  microscopical  examination  it  is  sufficient  to 
tease  out  a  piece  of  the  membrane  with  forceps  and  rub  it  on  a 
cover-glass,  or  if  it  be  somewhat  dry  a  small  drop  of  distilled 
water  should  be  added.  The  films  are  then  dried  in  the  usual 
way  and  stained  with  any  ordinary  basic  stain,  though  methylene- 
blue  is  on  the  whole  to  be  preferred,  used  either  as  a  saturated 
watery  solution  or  in  the  form  of  Loffler's  solution.  After 
staining  for  two  or  three  minutes  the  films  are  washed  in  water, 
dried,  and  mounted.  As  a  rule  no  decolorising  is  necessary,  as 


METHODS    OF   DIAGNOSIS.  375 

the  blue  does  not  overstain.  Neisser's  stain  (p.  363)  may  also 
be  used  with  advantage.  Any  secretion  from  the  pharynx  or 
other  part  is  to  be  treated  in  the  same  way.  The  value  of 
microscopical  examination  alone  depends  much  upon  the  experi- 
ence of  the  observer.  In  some  cases  the  bacilli  are  present  in 
characteristic  form  in  such  numbers  as  to  leave  no  doubt  in  the 
matter.  In  other  cases  a  few  only  may  be  found,  mixed  with 
large  quantities  of  other  organisms,  and  sometimes  their  charac- 
ters are  not  sufficiently  distinct  to  render  a  definite  opinion 
possible.  We  have  frequently  obtained  the  bacillus  by  means 
of  cultures,  when  the  result  of  microscopical  examination  of  the 
same  piece  of  membrane  was  non-conclusive.  As  already  said, 
however,  microscopical  examination  alone  is  more  reliable  after 
the  observer  has  had  experience  in  examining  cases  of  diphtheria 
and  making  cultures  from  them. 

(^)  By  making  Cultures.  —  For  this  purpose  a  piece  of  the 
membrane  should  be  separated  by  forceps  from  the  pharynx  or 
other  part  when  that  is  possible.  It  should  be  then  washed 
well  in  a  tube  containing  sterile  water,  most  of  the  surface  im- 
purities being  removed  in  this  way.  A  fragment  is  then  fixed 
in  a  platinum  loop  by  means  of  sterile  forceps,  and  a  series  of 
stroke-cultures  are  made  on  the  surface  of  any  of  the  media 
mentioned  (p.  361),  the  same  portion  of  the  membrane  being  al- 
ways brought  into  contact  with  the  surface.  The  tubes  are  then 
placed  in  the  incubator  at  37°  C,  and,  in  the  case  of  the  serum  me- 
dia and  blood  agar,  the  circular  colonies  of  the  diphtheria  bacillus 
are  visible  in  twenty-four  hours.  A  small  portion  of  a  colony  is 
then  removed  by  means  of  a  platinum  needle,  stained,  and  ex- 
amined in  the  usual  way,  the  characteristic  appearance  of  the 
organism  being  readily  recognised. 

In  cases  where  a  suspicion  arises  that  the  organism  found  is 
the  pseudo-diphtheria  bacillus,  bouillon  containing  a  trace  of 
glucose  should  be  inoculated  and  incubated  at  37°  C.  The  re- 
action should  be  tested  after  one  and  after  two  days'  growth. 
If  it  remains  alkaline,  the  diphtheria  bacillus  may  be  excluded. 
If  an  acid  reaction  results,  then  all  the  microscopical  and  cultural 
characters  must  be  carefully  observed,  and  the  virulence  of  the 
bacillus  may  be  ascertained  by  inoculating  a  guinea-pig,  say 
with  i  c.c.  of  a  broth  culture  of  two  days'  growth.  (See  also 
PP-  370,  37i,  372.) 


CHAPTER    XVII. 

TETANUS.1 

SYNONYMS.  —  LOCKJAW.     GERMAN,  WUNDSTARRKRAMPF. 
FRENCH,  TETANOS. 

Introductory. — Tetanus  is  a  disease  which  in  natural  conditions 
affects  chiefly  man  and  the  horse.  Clinically  it  is  characterised 
by  the  gradual  onset  of  general  spasms  of  the  voluntary  muscles, 
commencing  in  those  of  the  jaw  and  the  back  of  the  neck,  and 
extending  to  all  the  muscles  of  the  body.  These  spasms  are  of 
a  tonic  nature,  and,  as  the  disease  advances,  succeed  each  other 
with  only  a  slight  intermission  of  time.  There  are  often,  towards 
the  end  of  a  case,  fever  and  rise  of  respiration  and  pulse  rate. 
The  disease  is  usually  associated  with  a  wound  received  from 
four  to  fourteen  days  previously,  and  which  has  been  denied  by 
earth  or  dung.  Such  a  wound  may  be  very  small.  The  disease 
is,  in  the  majority  of  cases,  fatal.  Post  mortem  there  is  little  to 
be  observed  on  naked-eye  examination.  The  most  marked  fea- 
ture is  the  occurrence  of  patches  of  congestion  in  the  spinal 
cord,  and  especially  the  medulla. 

Historical.  —  The  general  association  of  the  development  of  tetanus  with 
the  presence  of  wounds,  though  these  might  be  very  small,  suggested  that 
some  infection  took  place  through  the  latter,  but  for  long  nothing  was  known 
as  to  the  nature  of  this  infection.  Carle  and  Rattone  in  1884  announced  that 
they  had  produced  the  disease  in  a  number  of  animals  by  inoculation  with 
material  from  a  wound  in  tetanus.  They  thus  demonstrated  the  trans- 
missibility  of  the  disease.  Nicolaier  (1885)  infected  mice  and  rabbits  with 
garden  earth,  and  found  that  many  of  them  developed  tetanus.  Suppuration 
occurred  in  the  neighbourhood  of  the  point  of  inoculation,  and  in  this  pus, 
besides  other  organisms,  there  was  always  present,  when  tetanus  had  occurred, 
a  bacillus  having  certain  constant  microscopic  characters.  Inoculation  of  fresh 
animals  with  such  pus  reproduced  the  disease.  Nicolaier^  attempts  at  its 
isolation  by  the  ordinary  gelatin  plate-culture  method  were,  however,  un- 

1  This  disease  is  not  to  be  confused  with  the  "  tetany  "  of  infants,  which  in  its 
essential  pathology  probably  differs  from  tetanus.  This  remark  of  course  does  not 
exclude  the  possibility  of  the  occurrence  of  true  tetanus  in  very  young  subjects. 

376 


BACILLUS   TETANI. 


377 


successful.  He  succeeded  in  getting  it  to  grow  in  liquid  blood  serum,  but 
always  in  mixture  with  other  organisms.  Infection  of  animals  with  such  a 
culture  produced  the  disease.  These  results  were  confirmed  by  Rosenbach, 
who,  though  failing  to  obtain  a  pure  culture,  cultivated  the  other  organisms 
present,  and  inoculated  them,  but  with  negative  results.  He  further  pointed 
out,  as  characteristic  of  the  bacillus,  its  development  of  terminal  spores.  In 
1889,  Kitasato  succeeded  in  isolating,  from  the  local  suppuration  of  mice  inoc- 
ulated from  a  human  case,  several  bacilli,  only  one  of  which,  when  injected  in 
pure  culture  into  animals,  caused  the  disease,  and  which  was  now  named  the 
B.  tetani.  This  organism  is  the  same  as  that  observed  by  Nicolaier  and  Rosen- 
bach.  Kitasato  found  that  the  cause  of  earlier  culture  failures  was  the  fact 
that  it  could  only  grow  in  the  absence  of  oxygen.  The  pathology  of  the 
disease  was  further  elucidated  by  Faber,  who,  having  isolated  bacterium-free 
poisons  from  cultures,  reproduced  the  symptoms  of  the  disease. 

Bacillus  Tetani.  —  If  in  a  case  of  tetanus  naturally  arising  in 
man  there  be  a  definite  wound  with  pus  formation  or  necrotic 
change,  the  bacillus  tetani  may  be  recognised  in  film  preparations 
from  the  pus, 

if   the   charac-  % 

teristic     spore  / 

formation    has 
occurred  (Fig.  -i 

126).     If;  how-  / 

ever,  the  teta- 
nus bacilli  have 
not  formed 
spores,  they  ap- 
pear as  some- 
what slen- 
der rods,  with- 
out presenting 
any  character- 
istic features. 
There  is  usu- 
ally present  in 
such  pus  a 
great  variety 
of  other  organ- 
isms  —  cocci 
and  bacilli. 

The  characters  of  the  bacillus  are,  therefore,  best  studied  in  cul- 
tures.    It  is  then  seen  to  be  a  slender  organism,  usually  about 


- 1   N' 


I 


r* 
" 


FlG.  126.  —  Film  preparation  of  discharge  from  wound  in  a  case 
of  tetanus,  showing  several  tetanus  bacilli  of  "  drumstick  "  form. 
(The  thicker  bacillus  with  oval  and  not  quite  terminal  spore,  in 
the  upper  part  of  the  field  towards  the  right  side,  is  not  a  tetanus 
bacillus  but  a  putrefactive  anaerobe  which  was  obtained  in  pure 
culture  from  the  wound.)  Stained  with  gentian-violet.  X  1000. 


378 


TETANUS. 


4  /*<  to  5  fji  in  length  and  .4  /A  in  thickness,  with  somewhat  rounded 

ends.  Besides  occurring  as  short  rods  it  also  develops  filamentous 

forms,  the  latter 

.%  being  more  com- 

mon in  fluid  me- 

A  **^  *  dia.  It  stains 

readily  by  any  of 
the  usual  stains 
and  also  by 
Gram's  method. 
A  feature  in  it 
is  the  uniform- 
ity with  which 
the  protoplasm 
stains.  It  is  very 
slightly  motile, 
and  its  motil- 
ity  can  be  best 
studied  in  an 
anaerobic  hang- 
ing-drop prepa- 
ration (p.  69). 

When  stained  by  the  special  methods  already  described,  it  is 

found  to  possess  numerous  delicate  flagella  attached  both  at 

the  sides  and  at  the  ends  (Fig. 

127).      These  flagella,  though 

they  may  be  of   considerable 

length,   are  usually  curled   up 

close  to  the  body  of  the  bacillus. 

The  formation  of  flagella  can 

be  best  studied  in  preparations 

made   from    surface   anaerobic 

cultures   (p.    66).      As   is   the 

case  with  many  other  anaerobic 

flagellated  bacteria  the  flagella, 

on    becoming   detached,   often 

become  massed  together  in  the 

form  of  spirals  of  striking  ap- 
pearance (Fig.  128).  At  incuba- 
tion temperature  B.  tetani  readily  forms  spores,  and  then  presents 


t 


FlG.  127.  —  Tetanus   bacilli,   showing  flagella. 
Muir's  method.     X  1000. 


Stained  by  Rd. 


FlG.  128.  —  Spiral  composed  of  numerous 
twisted  flagella  of  the  tetanus  bacillus.  Stained 
by  Rd.  Muir's  method.  X  1000. 


ISOLATION    OF   B.    TETANI. 


379 


a  very  characteristic  appearance.  The  spores  are  round,  and  in 
diameter  may  be  three  or  four  times  the  thickness  of  the  bacilli. 
They  are  developed  at  one  end  of  a  bacillus,  which  thus  assumes 
what  is  usually  described  as  the  drumstick  form  (Figs.  126, 
129).  Upon  rare  occasions  a  spore  may  form  at  each  pole  of 
a  bacillus,  producing  a  dumb-bell  form  (see  Fig.  130).  In  a  speci- 
men stained  with  a  watery  solution  of  gentian-violet  or  methy- 


FlG.  129.  —  Tetanus  bacilli;  some  of 
which  possess  spores.  From  a  culture  in 
glucose  agar,  incubated  for  three  days  at 
37°  C.  Stained  with  carbol-fuchsin.  x  1000. 


FIG.  130.  —  Bipolar  spore  formation  in 
a  glucose  agar  culture  of  B.  tetani.  (Dr. 
Chas.  H.  Potter.)  x  1000. 


lene-blue,  the  spores  are  uncoloured  except  at  the  periphery,  so 
that  the  appearance  of  a  small  ring  is  produced ;  if  a  powerful 
stain  such  as  carbol-fuchsin  be  applied  for  some  time,  the  spores 
become  deeply  coloured  like  the  bacilli.  Further,  especially  if 
the  culture  preparation  be  heated,  the  spores  may  become  free 
in  the  culture  medium. 

Isolation.  — The  isolation  of  the  tetanus  bacillus  is  somewhat 
difficult.  By  inoculation  experiments  in  animals,  its  natural 
habitat  has  been  proved  to  be  garden  soil,  and  especially  the 
contents  of  dung  heaps,  where  it  probably  leads  a  saprophytic 
existence,  though  its  function  as  a  saprophyte  is  unknown.  From 
such  sources  and  from  the  pus  of  wounds  in  tetanus,  occurring 
naturally  or  experimentally  produced,  it  has  been  isolated  by 
means  of  the  methods  appropriate  for  anaerobic  bacteria.  The 
best  methods  for  dealing  with  such  pus  are  as  follows  :  - 

(i)  The  principle  is  to  take  advantage  of  the  resistance  of 
the  spores  of  the  bacillus  to  heat.  A  sloped  tube  of  inspissated 


380  TETANUS. 

serum  or  a  deep  tube  of  glucose  agar  is  inoculated  with  the  pus 
and  incubated  at  37°  C.  for  forty-eight  hours,  at  the  end  of 
which  time  numerous  spore-bearing  bacilli  can  often  be  observed 
microscopically.  The  culture  is  then  kept  at  80°  C.  for  from 
three-quarters  to  one  hour,  with  the  view  of  killing  all  organisms 
except  those  which  have  spored.  A  loopful  is  then  added  to 
glucose  gelatin,  and  roll-tube  cultures  are  made  in  the  usual 
way  and  kept  in  an  atmosphere  of  hydrogen  at  22°  C.  ;  after 
five  days  the  plates  are  ready  for  examination.  Kitasato  com- 
pares the  colonies  in  gelatin  plates  to  those  of  the  B.  subtilis. 
They  consist  of  a  thick  centre  with  shoots  radiating  out  on  all 
sides.  They  liquefy  the  gelatin  more  slowly  than  the  B. 
subtilis.  This  method  of  isolation  is  not  always  successful, 
partly  because  along  with  the  tetanus  bacilli,  both  in  its  natural 
habitats  outside  the  body  and  in  the  pus  of  wounds,  other  spore- 
forming  obligatory  and  facultative  anaerobes  occur,  which  grow 
faster  than  the  tetanus  bacillus,  and  thus  overgrow  it. 

(2)  If  in  any  discharge  the  spore-bearing  tetanus  bacilli  be 
seen   on  microscopic  examination,  then  a  method  of  isolation 
based  on  the  same  principle  as  the  last  may  be  adopted.    Inocu- 
lations with  the  suspected  material  are  made  in  half  a  dozen 
deep  tubes  of  glucose  agar,  previously  melted  and  kept  at  a 
temperature    of    100°    C.      After    inoculation    they    are    again 
placed  in  boiling  water  and  kept  for  varying  times,  say  for  half 
a  minute,  for  one,  three,  four,  five,  and  six  minutes  respectively. 
They  are  then  plunged  in  cold  water  till  cool,  and  thereafter 
placed  in  the  incubator  at  37°  C.,  in  the  hope  that  in  one  or 
other  of  the  tubes   all  the  organisms  present  will  have  been 
killed,  except  the  tetanus   spores  which   can   develop  in  pure 
culture. 

(3)  Some  method  of  anaerobically  making  plates,  such  as 
that  of  Bulloch  or  Novy,  may  be  employed.     The  isolation  of 
the  tetanus  bacillus  is  in  many   cases  a  difficult  matter,  and 
various  expedients  require  to  be  tried. 

Characters  of  Cultures.  — -Pure  cultures  having  been  obtained, 
sub-cultures  can  be  made  in  deep  upright  glucose  gelatin  or  agar 
tubes.  On  glucose  gelatin  in  such  a  tube  there  commences,  an 
inch  or  so  below  the  surface,  a  growth  consisting  of  fine  straight 
threads,  rather  longer  in  the  lower  than  in  the  upper  parts  of  the 
tube,  radiating  out  from  the  needle  track  (Fig.  131).  Slow  lique- 


PATHOGENIC   EFFECTS.  381 

faction  of  the  gelatin  takes  place,  with  slight  gas  formation.  In 
agar  the  growth  is  somewhat  similar,  consisting  of  small  nodules 
along  the  needle  track,  with  irregular  short  offshoots  passing 
out  into  the  medium  (Fig.  134,  A).  There  is 
slight  formation  of  gas,  but,  of  course,  no  lique- 
faction. Growth  also  occurs  in  blood  serum  and 
also  in  glucose  bouillon  under  anaerobic  condi- 
tions. The  latter  is  the  medium  usually  employed 
for  obtaining  the  soluble  products  of  the  organ- 
ism. There  is  in  it  at  first  a  slight  turbidity,  and 
later  a  thin  layer  of  a  powdery  deposit  on  the  walls 
of  the  vessel.  All  the  cultures  give  out  a  peculiar 
burnt  odour  of  rather  unpleasant  character. 

Conditions  of  Growth,  etc.  —  The  B.  tetani 
grows  best  at  37°  C.  The  minimum  growth  tem- 
perature is  about  14°  C.,  and  below  22°  C.  growth 
takes  place  very  slowly.  Growth  takes  place  only 
in  the  absence  of  oxygen,  the  organism  being  a 
strict  anaerobe.  Sporulation  may  commence  at 
the  end  of  twenty-four  hours  in  cultures  grown 
at  37°  C.,  —  much  later  at  lower  temperatures. 
Like  other  spores,  those  of  tetanus  are  extremely 
resistant.  They  can  usually  withstand  boiling  for 
five  minutes,  and  can  be  kept  in  a  dry  condition 
for  many  months  without  being  killed  or  losing 
their  virulence.  They  have  also  high  powers  of  culture*  of' The^te- 
resistance  to  antiseptics.  tanus  bacillus  in 

Pathogenic  Effects. —The  proof  that  the  B.  sho^Sg thejtS^i 
tetani  is  the  cause  of  tetanus  is  complete.     It  can  shoots  (Kitasato). 

....  ,       ,  .     .  ,     Natural  size. 

be  isolated  in  pure  culture,  and  when  re-injected 
in  pure  culture  it  reproduces  the  disease.  It  may  be  impossible 
to  isolate  it  from  some  cases  of  the  disease,  but  the  cause  of  this 
very  probably  is  the  small  numbers  in  which  it  sometimes  occurs. 
(a)  The  Disease  as  arising  Naturally.  —  The  disease  occurs 
naturally,  chiefly  in  horses  and  in  man.  Other  animals  may, 
however,  be  affected.  There  is  usually  some  wound,  often  of  a 
ragged  character,  which  has  either  been  made  by  an  object 
soiled  with  earth  or  dung,  or  which  has  become  contaminated 
with  these  substances.  There  is  often  purulent  or  foetid  dis- 
charge, though  this  may  be  absent.  Microscopic  examination 


382  TETANUS. 

of  sections  may  show  at  the  edges  of  the  wound  necrosed  tis- 
sue in  which  the  tetanus  bacilli  may  be  very  numerous.  If  a. 
scraping  from  the  wound  be  examined  microscopically,  bacilli 
resembling  the  tetanus  bacillus  may  be  recognised.  If  these 
have  spored,  there  can  be  practically  no  doubt  as  to  their 
identity,  as  the  drumstick  appearance  which  the  terminal  spore 
gives  to  the  bacillus  is  not  common  among  other  bacilli.  Care 
must  be  taken,  however,  to  distinguish  it  from  other  thicker 
bacilli  with  oval  spores  placed  at  a  short  distance  from  their 
extremities,  such  forms  being  common  in  earth,  etc.,  and  also 
met  with  in  contaminated  wounds  (Fig.  126).  It  is  important  to 
note  that  the  wound  through  which  infection  has  taken  place 
may  be  very  small,  in  fact,  may  consist  of  a  mere  abrasion.  In 
some  cases,  especially  in  the  tropics,  it  may  be  merely  the  bite 
of  an  insect.  The  absence  of  a  definite  channel  of  infection 
has  given  rise  to  the  term  "idiopathic  "  tetanus.  There  is,  how- 
ever, practically  no  doubt  that  all  such  cases  are  true  cases  of 
tetanus,  and  that  in  all  of  them  the  cause  is  the  B.  tetani.  The 
latter  has  also  been  found  in  the  bronchial  mucous  membrane 
in  some  cases  of  the  so-called  rheumatic  tetanus,  the  cause  of 
which  is  usually  said  to  be  cold. 

The  pathological  changes  found  post  mortem  are  not  striking. 
There  may  be  haemorrhages  in  the  muscles  which  have  been  the 
subject  of  the  spasms.  These  are  probably  due  to  mechanical 
causes.  Naturally  it  is  in  the  nervous  system  that  we  look  for 
the  most  important  lesions.  Here  there  is  ordinarily  a  general 
redness  of  the  grey  matter,  and  the  most  striking  feature  is  the 
occurrence  of  irregular  patches  of  slight  congestion  which  are 
not  limited  particularly  to  grey  or  white  matter,  or  to  any  tract 
of  the  latter.  These  patches  are  usually  best  marked  in  the 
grey  matter  of  the  medulla  and  pons.  Microscopically  there  is. 
little  of  a  definite  nature  to  be  found.  There  is  congestion,  and 
there  may  be  minute  haemorrhages  in  the  areas  noted  by  the 
naked  eye.  The  ganglion  cells  may  show  appearances  which 
have  been  regarded  as  degenerative  in  nature,  and  similar 
changes  have  been  described  in  the  white  matter.  The  only 
marked  feature  is  thus  a  vascular  disturbance  in  the  central 
nervous  system,  with  a  possible  tendency  to  degeneration  in  its 
specialised  cell.  Both  of  these  conditions  are  probably  due  to 
the  action  of  the  toxins  of  the  bacillus.  In  the  case  of  the 


EXPERIMENTAL    INOCULATION.  383 

cellular  degenerations  the  cells  have  been  observed  to  return 
to  the  normal  under  the  curative  influence  of  the  antitoxins 
(vide  infra).  In  the  other  organs  of  the  body  there  are  no 
constant  changes. 

We  have  said  that  the  general  distribution  of  pathogenic 
bacteria  throughout  the  body  is  probably  a  relative  phenomenon, 
and  that  bacteria  usually  found  locally  may  occur  generally,  and 
vice  versa.  With  regard  to  the  tetanus  bacillus  it  is,  however, 
probably  the  case  that  very  rarely,  if  ever,  are  the  organisms 
found  anywhere  except  in  the  local  lesion. 

(b)  The  Artificially-produced  Disease. — The  disease  can  be 
communicated  to  animals  by  any  of  the  usual  methods  of  inocu- 
lation, but  does  not  arise  in  animals  fed  with  bacilli,  whether 
these  contain  spores  or  not.  Kitasato  found  that  pure  cultures, 
injected  subcutaneously  or  intravenously,  caused  death  in  mice, 
rats,  guinea-pigs,  and  rabbits.  In  mice,  symptoms  appear  in  a 
day,  and  death  occurs  in  two  or  three  days,  after  inoculation 
with  a  loopful  of  a  bouillon  culture.  The  other  animals  men- 
tioned require  larger  doses,  and  death  does  not  occur  so  rapidly. 
Usually  in  animals  injected  subcutaneously  the  spasms  begin 
in  the  limb  nearest  the  point  of  inoculation.  In  intravenous 
inoculation  the  spasms  begin  in  the  extensor  muscles  of  the 
trunk,  as  is  the  case  in  the  natural  disease  in  man.  After  death 
there  is  found  slight  hyperaemia,  without  pus  formation,  at  the 
seat  of  inoculation.  The  bacilli  diminish  in  number,  and  may 
be  absent  at  the  time  of  death.  The  organs  generally  show 
little  change. 

Kitasato  states  that  in  his  earlier  experiments  the  quantity  of 
culture  medium  injected  along  with  the  bacilli  already  contained 
enough  of  the  poisonous  bodies  formed  by  the  bacilli  to  cause 
death.  The  symptoms  came  on  sooner  than  by  the  improved 
method  mentioned  below,  and  were,  therefore,  due  to  the  toxins 
already  present.  In  his  subsequent  work,  therefore,  he  em- 
ployed splinters  of  wood  soaked  in  cultures  in  which  spores 
were  present,  and  subsequently  subjected  for  one  hour  to  a 
temperature  of  80°  C.  The  latter  treatment  not  only  killed  all 
the  bacilli,  but,  as  we  shall  see,  was  sufficient  to  destroy  the 
activity  of  the  toxins.  When  such  splinters  are  introduced 
subcutaneously,  death  results  by  the  development  of  the  spores 
which  they  carry.  In  this  way  he  completed  the  proof  that 


384  TETANUS. 

the  bacilli  by  themselves  can  form  toxins  in  the  body  and  pro- 
duce the  disease.  Further,  if  a  small  quantity  of  garden  earth 
be  placed  under  the  skin  of  a  mouse,  or  better  that  of  a  white 
rat,  death  from  tetanus  takes  place  in  a  great  many  cases. 
[Sometimes,  however,  in  such  circumstances  death  occurs  with- 
out tetanic  symptoms,  and  is  not  due  to  the  tetanus  bacillus  but 
to  the  bacillus  of  malignant  oedema,  which  also  is  of  common 
occurrence  in  the  soil  (vide  infra).']  By  such  experiments,  sup- 
plemented by  the  culture  experiments  mentioned,  the  natural 
habitats  of  the  B.  tetani,  as  given  above,  have  become  known. 

The  Toxins  of  the  Tetanus  Bacillus.  —  The  tetanus  bacillus 
being  thus  accepted  as  the  cause  of  the  disease,  we  have  to 
consider  how  it  produces  its  pathogenic  effects. 

Almost  contemporaneously  with  the  work  on  diphtheria  was  the  attempt 
made  with  regard  to  tetanus  to  explain  the  general  symptoms  by  supposing 
that  the  bacillus  could  excrete  soluble  poisons.  The  earlier  results  in  which 
certain  bases,  tetanin  and  tetanotoxin,  were  said  to  have  been  isolated,  have 
only  a  historic  interest,  as  they  were  obtained  by  faulty  methods.  In  1890 
Brieger  and  Fraenkel  announced  that  they  had  isolated  a  toxalbumin  from 
tetanus  cultures,  and  this  body  was  independently  discovered  by  Faber  in  the 
same  year.  Brieger  and  Fraenkel's  body  consisted  practically  of  an  alcoholic 
precipitate  from  filtered  culture  in  bouillon,  and  was  undoubtedly  toxic. 
Within  recent  years  such  attempts  to  isolate  tetanus  toxins  in  a  pure  condi- 
tion have  practically  been  abandoned,  and  attention  has  been  turned  to  the 
investigation  of  the  physiological  effects  either  of  the  crude  toxin  present  in 
filtered  bouillon  cultures,  or  of  the  precipitate  produced  from  the  same  by 
ammonium  sulphate  (cf.  p.  176). 

The  toxic  properties  of  bacterium-free  filtrates  of  pure  cultures 
of  the  B.  tetani  were  investigated  in  1891  by  Kitasato.  This 
observer  found  that  when  the  filtrate,  in  certain  doses,  was 
injected  subcutaneously  or  intravenously  into  mice,  tetanic 
spasms  developed,  first  in  muscles  contiguous  to  the  site  of 
inoculation  and  later  all  over  the  body.  Death  resulted.  He 
found  that  guinea-pigs  were  more  susceptible  than  mice,  and 
rabbits  less  so.  In  order  that  a  strongly  toxic  bouillon  be  pro- 
duced, it  must  originally  have  been  either  neutral  or  slightly 
alkaline.  Kitasato  further  found  that  the  toxin  was  easily  in- 
jured by  heat.  Exposure  for  a  few  minutes  at  65°  C.  destroyed 
it.  It  was  also  destroyed  by  twenty  minutes'  exposure  at  60°  C., 
and  by  one  and  a  half  hours'  at  55°  C.  Drying  had  no  effect. 
It  was,  however,  destroyed  by  various  chemicals  such  as  pyro- 


TOXINS    OF   THE   TETANUS    BACILLUS.  385 

gallol  and  also  by  sunlight.  Behring  has  more  recently  pointed 
out  that  after  the  nitration  of  cultures  containing  toxin,  the  lat- 
ter may  very  rapidly  lose  its  power,  and  in  a  few  days  may  only 
possess  yj-ffth  of  its  original  toxicity.  This  he  attributes  to  such 
factors  as  temperature  and  light,  and  especially  to  the  action  of 
oxygen.  The  effect  of  these  agents  on  the  crude  toxin  is  un- 
doubtedly to  cause  a  degeneration  of  the  true  toxin  into  a  series 
of  toxoids  similar  to  those  produced  in  the  case  of  diphtheria 
toxin,  and  it  is  also  true  here  that  the  toxoids  while  losing  their 
toxicity  may  still  retain  their  power  of  producing  immunity 
against  the  potent  toxin.  Further,  altogether  apart  from  the 
occurrence  side  by  side  in  the  crude  toxin  of  strong  and  weak 
poisons,  it  has  been  shown  that  such  crude  toxin  contains  toxic 
substances  of  probably  quite  a  different  nature.  Ehrlich  has 
shown  that  besides  the  predominant  spasm-producing  toxin 
(called  by  him  tetanospasmin),  there  exists  in  crude  toxin  a 
poison  capable  of  producing  the  solution  of  certain  red  blood 
corpuscles.  This  hsemolytic  agent  he  calls  tetanolysin.  It 
does  not  occur  in  all  samples  of  crude  tetanus  toxin,  nor  is  it 
found  when  a  bouillon  culture  of  the  bacillus  is  filtered  through 
porcelain.  To  obtain  it  the  fresh  culture  must  be  treated  by 
ammonium  sulphate,  as  described  in  the  method  of  obtaining 
concentrated  toxins  (p.  176).  This  substance  also  has  the  power 
of  originating  an  antitoxin  so.  that  certain  antitetanic  sera  can 
protect  red  blood  corpuscles  against  its  action.  Madsen,  study- 
ing the  interactions  of  this  antitetanolysin  with  the  tetanolysin, 
has  shown  that  the  latter  may  appear  in  the  form  of  an  active 
poison,  and  of  bodies  corresponding  to  toxoids,  and  he  has  con- 
firmed Ehrlich's  views  on  the  possession  by  true  toxins  of 
haptophorous  and  toxophorous  groups.  That  there  are  close 
resemblances  in  nature  between  the  tetanus  and  diphtheria  tox- 
ins is  further  shown  by  the  fact  that  the  action  of  an  acid  on 
tetanus  toxin  is  to  cause  an  apparent  disappearance  of  toxicity, 
but  if  before  a  certain  time  has  elapsed  the  acid  be  neutralised 
by  alkali,  then  a  degree  of  the  toxicity  returns. 

Various  attempts  have  been  made  to  find  out  the  nature  of 
the  tetanus  poisons.  Sidney  Martin  derived  from  the  organs  of 
persons  dead  of  tetanus  two  classes  of  bodies.  One  of  these 
consisted  of  a  purified  alcoholic  precipitate  (formed  chiefly  of 
albumoses).  To  these  he  attributes  a  fever-producing  action. 

2C 


386  TETANUS. 

The  other  bodies  were  those  soluble  in  alcohol  and  also  in  ether. 
They  were  non-proteid,  and  to  them  he  attributed  the  excitation 
of  the  muscular  spasms  in  tetanus.  Uschinsky,  moreover,  has 
found  that  the  bacillus  can  produce  its  toxin  when  growing  in  a 
fluid  containing  no  proteid  matter.  The  toxin  may  thus  be 
formed  independently  of  the  breaking  up  of  the  proteids  on 
which  the  bacillus  may  be  living,  though  it  no  doubt  has  a 
digestive  action  on  these.  Brieger  also  now  apparently  thinks 
that  the  toxicity  of  the  toxalbumins  originally  described  by  him 
is  due  to  the  presence  of  a  non-proteid  body. 

It  is  thought  by  some  that  a  diastase  is  concerned  in  the  toxic 
action  of  the  tetanus  bacillus.  Like  a  ferment,  the  toxin  is  de- 
stroyed, as  we  have  seen,  by  comparatively  low  temperatures, 
but,  as  has  already  been  pointed  out  (Chap.  VI.),  it  may  simply 
be  an  unstable  chemical  compound,  for  albuminous  bodies  not 
diastatic  in  nature  may  be  changed  at  similar  temperatures. 
The  liquefaction  (i.e.  probable  peptonisation)  of  gelatin  cultures 
advances  pan  passu  with  the  development  of  toxins,  and  filtered 
bacterium-free  cultures  will  still  liquefy  gelatin.  It  is  probable, 
however,  that  there  is  developed,  in  addition,  a  peptic  ferment 
which  will,  of  course,  also  pass  through  the  filter.  For  if  equal 
portions  of  the  filtered  culture  be  left  in  contact  with  equal  por- 
tions of  gelatin  for  various  lengths  of  time,  there  is  no  increase 
of  toxicity  in  those  kept  longest.  There  is  thus  no  fresh  devel- 
opment of  toxin  during  the  advancing  liquefaction  of  the  gelatin. 
Thus  peptic  digestion  and  toxic  formation  are  apparently  due  to 
different  vital  processes  on  the  part  of  the  tetanus  bacillus. 

An  argument  in  favour  of  a  ferment  being  concerned  in  the 
toxin  production  is  derived  from  the  occurrence  of  a  definite 
incubation  period  between  the  introduction  of  the  toxin  into  an 
animal's  body  and  the  appearance  of  symptoms.  The  incubation 
period  varies  according  to  the  species  of  animal  employed,  and 
the  path  of  infection.  In  the  guinea-pig  it  is  from  thirteen  to 
eighteen  hours,  in  the  horse  five  days,  and  the  incubation  is 
shorter  when  the  poison  is  introduced  into  a  vein  than  when 
injected  subcutaneously.  The  interpretation  put  on  the  occur- 
rence of  this  period  of  incubation  by  the  upholders  of  the  fer- 
ment theory  has  been  that  a  time  is  required  for  the  supposed 
diastase  to  elaborate  from  the  tissues  albumoses,  which  are  the 
immediately  toxic  agents. 


ACTION    OF   THE   TOXIN.  387 

Whatever  the  nature  of  the  toxin  is,  it  is  undoubtedly  one 
of  the  most  powerful  poisons  known.  Even  with  his  probably 
impure  toxalbumin  Brieger  found  that  the  fatal  dose  for  a  mouse 
was  .0005  of  a  milligramme.  If  the  susceptibility  of  man  be 
the  same  as  that  of  a  mouse,  the  fatal  dose  for  an  average  adult 
would  be  .23  of  a  milligramme  or  about  2^ootns  °f  a  grain. 

With  regard  to  the  action  of  the  toxin  it  has  been  shown  to 
have  no  effect  on  the  sensory  or  motor  endings  of  the  nerves. 
It  acts  solely  as  an  exciter  of  the  reflex  excitability  of  the  motor 
cells  in  the  spinal  cord.  The  motor  cells  in  the  pons  and 
medulla  are  also  affected,  and  to  a  much  greater  degree  than 
those  in  the  cerebral  cortex.  When  injected  subcutaneously 
the  toxin  probably  to  a  certain  extent  is  absorbed  into  the 
sheaths  of  the  nerves,  and  thence  finds  its  way  to  that  part  of 
the  spinal  cord  from  which  these  nerves  spring.  This  explains 
the  fact  that  in  an  animal  often  the  tetanic  spasms  appear  first 
in  the  muscles  of  the  part  in  which  the  inoculation  has  taken 
place.  It  is  doubtful  whether  such  absorption  takes  place  in 
tetanus  arising  naturally  in  man.  In  artificial  injection  of  toxin 
part  finds  its  way  into  the  blood  stream,  and  if  infected  animals 
be  killed  during  the  incubation  period  there  is  often  evidence 
of  toxin  in  the  blood  and  solid  organs.  Rarely,  however,  during 
this  period,  and  probably  never  after  symptoms  have  begun,  is 
there  free  toxin  in  the  central  nervous  system.  In  the  guinea- 
pig  there  is  little  doubt  that  tetanus  toxin  has  an  affinity  solely 
for  the  nervous  system.  In  other  animals,  such  as  the  rabbit, 
an  affinity  may  exist  in  other  organs,  and  the  fixation  of  the 
poison  in  such  situations  may  give  rise  to  no  recognisable  symp- 
toms. In  such  an  animal  as  the  alligator,  it  is  possible  that 
while  some  of  its  organs  have  an  affinity  for  tetanus  toxin  its 
nervous  system  has  none.  These  facts  are  of  great  scientific 
interest,  and  a  possible  explanation  of  them  will  be.  discussed  in 
the  chapter  on  Immunity.  If  tetanus  toxin  be  introduced  into 
the  stomach  or  intestine,  it  is  not  absorbed.  It  to  a  large  extent 
passes  through  the  intestine  unchanged.  Evidence  that  any 
destruction  takes  place  is  wanting. 

Toxin  in  the  Circulating  Blood.  —  A  research  .of  far-reaching  importance 
upon  the  amount  of  tetanus  toxin  circulating  in  the  blood  of  horses  infected 
with  the  disease  has  just  been  completed  by  Bolton  and  Fisch  of  St.  Louis. 
It  was  undertaken  as  a  result  of  an  inquiry  into  a  number  of -fatal  cases  of  teta- 


388  TETANUS. 

nus  developing  in  diphtheria  patients  who  were  being  treated  with  antitoxin. 
Evidence  was  brought  forth  which  showed  that  a  horse  supplying  the  antitoxic 
serum  had  died  of  tetanus  shortly  after  the  bleeding  which  furnished  the  fatal 
serum,  but  at  the  time  of  the  bleeding  no  symptoms  of  tetanus  had  been  notice- 
able. Samples  of  this  serum  proved  to  contain  enough  tetanus  toxin  in  o.i  c.c. 
to  kill  a  guinea-pig  in  a  few  days. 

With  these  facts  before  them,  Bolton  'and  Fisch  carried  out  a  study  upon 
five  horses,  three  of  which  were  inoculated  artificially  with  B.  tetani,  and  two 
in  which  tetanus  had  developed  accidentally.  Summarized,  their  results  show 
that :  — 

1.  Tetanus  toxin  may  appear  in  the  blood  of  infected  horses  four  to  five  days 

before  symptoms  of  tetanus  are  manifested,  and  sufficient  to  kill  human 
beings  in  doses  of  10-100  c.c.  of  such  a  serum. 

2.  Within  a  day  or  two  of  the  death  of  an  infected  horse,  having  some  or  no 

symptoms  of  tetanus,  the  toxin  reaches  a  maximum  and  then  rapidly  de- 
clines, so  that  shortly  before  death  no  toxin  can  be  demonstrated. 

3.  Shortly  before  death  circulating  toxin  is  replaced  by  antitoxin. 

4.  It  is  quite  a  difficult  matter  to  induce  tetanus  in  the  horse  by  artificially 

inoculating  it  with  tetanus-bearing  earth. 

In  the  light  of  the  above  results,  which  are  decidedly  impressive,  it  is  now 
imperative,  for  the  preservation  of  the  public,  that  all  diphtheria  antitoxic 
serum  shall  be  tested  for  tetanus  toxin  before  being  marketed,  whether  horses 
furnishing  the  serum  appeared  to  be  in  health  or  not. 

There  is  one  question  which  must  arise  in  connection  with 
tetanus,  namely  :  Granted  that  the  B.  tetani  is  so  widely  present 
in  the  soil,  how  is  it  that  the  disease  is  not  more  common  than 
it  is,  for  wounds  must  constantly  be  contaminated  with  such 
soil  ?  Experiments  by  Vaillard  throw  light  on  this  point.  We 
have  seen  that  unless  suitable  precautions  are  adopted,  in  exper- 
imental tetanus  in  animals  death  results  not  from  inoculation 
but  from  an  intoxication  with  toxin  previously  existent  in  the 
fluid  in  which  the  bacilli  have  been  growing.  According  to 
Vaillard,  if  spores  rendered  toxin-free  by  being  kept  for  a  suf- 
ficient time  at  80°  C.  are  injected  into  an  animal,  death  does 
not  take  place.  It  was  found,  however,  that  such  spores  can  be 
rendered  pathogenic  by  injecting  along  with  them  such  chemicals 
as  lactic  acid,  by  injuring  the  point  of  inoculation  so  as  to  cause 
effusion  of  blood,  by  fracturing  an  adjacent  bone,  by  introduc- 
ing a  mechanical  irritant  such  as  soil  or  a  splinter  of  wood  (as 
in  Kitasato's  experiments),  or  by  the  simultaneous  injection  of 
other  bacteria  such  as  the  staphylococcus  pyogenes  aureus.  These 
facts,  especially  the  last,  throw  great  light  on  the  disease  as  it 
occurs  naturally,  for  tetanus  results  especially  from  wounds 


IMMUNITY   AGAINST   TETANUS.  389 

which  have  been  accidentally  subjected  to  conditions  such  as 
those  enumerated.  Kitasato  now  holds  that  in  the  natural 
infection  in  man,  along  with  tetanus  spores,  the  presence  of 
foreign  material  or  of  other  bacteria  is  necessary.  Spores  alone 
or  tetanus  bacilli  without  spores  die  in  the  tissues,  and  tetanus 
does  not  result. 

Summary.  —  In  view  of  all  the  facts  available  we  must  thus 
look  on  tetanus  as  caused  by  the  B.  tetani.  The  bacillus  gains 
entrance  to  the  body  through  wounds  or  abrasions,  and,  multi- 
plying locally,  produces  poisons  which  diffuse  into  the  tissues 
and  have  an  elective  action  as  stimulators,  especially  of  the 
spinal  cord.  The  chemical  composition  of  these  poisons  is  not 
yet  fully  known.  The  enormous  potency  of  such  poisons  explains 
how,  even  in  a  fatal  case,  extreme  smallness  of  the  wound  and 
difficulty  in  isolating  the  bacillus  do  not  detract  from  the  theory 
that  the  latter  is  the  cause  of  the  disease. 

Immunity  against  Tetanus.  —  Antitetanic  Serum.  —  The  arti- 
ficial immunisation  of  animals  against  tetanus  has  received 
much  attention.  The  most  complete  study  of  the  question  is 
found  in  the  work  of  Behririg  and  Kitasato  in  Germany,  and  of 
Tizzoni  and  Cattani  in  Italy.  The  former  observers  found  that 
such  an  immunity  could  be  conferred  by  the  injection  of  very 
small  and  progressively  increasing  doses  of  the  tetanus  toxin. 
The  degree  of  immunity  attained,  however,  was  not  high. 
More  successful  was  the  method  of  accompanying  the  early 
injections  of  such  toxin  with  the  subcutaneous  introduction  of 
small  doses  of  iodine  terchloride.  Tizzoni  and  Cattani  have  also 
used  the  method  of  administering  progressively  increasing  doses 
of  living  cultures  attenuated  in  various  ways,  e.g.  by  heat.  By 
any  of  these  methods  susceptible  animals  can  rapidly  acquire 
great  immunity,  not  only  against  many  times  the  fatal  dose  of 
tetanic  toxin,  but  also  against  injections  of  the  living  bacilli.  The 
degree  of  immunisation  acquired  by  an  animal  remains  in  exist- 
ence for  several  months.  Not  only  so,  but  when  a  high  degree 
of  immunity  has  been  produced  by  prolonged  treatment,  it  is 
found  that  the  serum  of  immune  animals  usually  possesses  the 
capacity,  when  injected  into  animals  susceptible  to  the  disease, 
of  protecting  them  against  a  subsequent  infection  with  a  fatal 
dose  of  tetanus  bacilli  or  toxin.  Further,  if  injected  subse- 
quently to  such  infection,  the  serum  can  in  certain  cases  prevent 


390  TETANUS. 

a  fatal  result,  even  when  symptoms  have  begun  to  appear.  The 
degree  of  success  attained  depends,  however,  on  the  shortness 
of  the  time  which  has  elapsed  between  the  infection  with  the 
bacilli  or  toxin  and  the  injection  of  the  serum.  The  longer 
the  interval  which  is  allowed  to  elapse,  not  only  the  greater 
must  be  the  dose  of  the  serum  but  the  less  likely  is  cure  to 
occur.  In  animals  where  symptoms  have  fully  manifested 
themselves  only  a  small  proportion  of  cases  can  be  saved.  As 
in  other  cases,  there  is  no  evidence  that  the  antitetanic  serum 
has  any  detrimental  effect  on  the  bacilli.  It  only  neutralises  the 
effects  of  the  toxin.  The  standardisation  of  the  antitetanic 
serum  is  of  the  highest  importance.  Behring  recommends 
that  for  protecting  animals  a  serum  should  be  obtained  of  which 
one  gramme  will  protect  1,000,000  grammes'  weight  of  mice 
against  the  minimum  fatal  dose  of  the  bacillus  or  toxin.  A 
mouse  weighing  twenty  grammes  would  thus  require  .00002 
gramme  of  such  a  serum  to  protect  it  against  the  minimum 
lethal  dose.  In  the  injection  of  such  a  serum  subsequent  to 
infection,  if  symptoms  have  begun  to  appear,  1000  times  this 
dose  would  be  necessary;  a  few  hours  later  10,000  times,  and 
so  on. 

As  the  result  of  his  experiments,  Behring  aimed  at  obtaining 
a  curative  effect  in  the  natural  disease  occurring  in  man.  For 
this  purpose,  as  for  his  later  laboratory  experiments,  he  obtained 
serum  by  the  immunisation  of  such  large  animals  as  the  horse, 
the  sheep,  and  the  goat.  The  principles  of  the  process  were 
the  same  as  in  his  earlier  work,  namely,  the  injection  of  toxin, 
accompanied  at  first  with  the  injection  of  iodine  terchloride.  It 
was  found  that  the  greater  the  degree  of  the  natural  susceptibility 
of  an  animal  to  tetanus,  the  easier  it  was  to  obtain  a  serum  of  a 
high  antitetanic  potency.  The  horse  was,  therefore,  the  most 
suitable  animal.  If  now  we  take  for  granted  that  the  relative 
susceptibility  of  man  and  the  mouse  towards  tetanus  are  nearly 
equal,  a  man  weighing  100  kilogrm.  would  require  .1  grm.  of 
the  serum  mentioned  above  to  protect  him  from  inoculation 
with  the  minimun  lethal  dose  of  bacilli  or  toxin.  If  symptoms 
had  begun  to  appear,  100  c.c.  at  once  would  be  necessary,  and 
as  the  injection  of  such  a  quantity  might  be  inconvenient, 
Behring  recommended  that  for  man  a  more  powerful  serum 
should  be  obtained,  viz.,  a  serum  of  which  one  gramme  would 


IMMUNITY   AGAINST   TETANUS.  391 

protect  100,000,000  grammes'  weight  of  mice.1  The  potency 
is  maintained  for  several  months  if  precautions  are  taken  to 
avoid  putrefaction,  exposure  to  bright  light,  etc.  To  this  end 
.5  per  cent  carbolic  acid  is  usually  added,  and  the  serum  is 
kept  in  the  dark.  In  a  case  of  tetanus  in  man,  100  c.c. 
of  such  a  serum  should  be  injected  within  twenty-four  hours 
in  five  doses,  each  at  a  different  part  of  the  body,  and 
this  followed  up  by  further  injections  if  no  improvement  takes 
place.  It  is  stated  that  better  results  have  been  obtained 
when  the  fluid  serum  has  been  injected  intravenously,  and  that 
large  amounts  can  be  safely  employed.  Of  this  method  we 
have  had  no  experience.  The  serum  has  also  been  introduced 
intracerebrally,  very  slow  injection  into  the  brain  substance 
being  practised,  but  no  better  results  have  been  obtained  than 
by  the  subcutaneous  method. 

Many  cases  of  human  tetanus  have  been  thus  treated,  but 
with  only  a  small  measure  of  success.  The  improvement  in 
the  death-rate  has  not  been  nearly  so  marked  as  that  which  has 
occurred  in  diphtheria,  under  similar  circumstances.  As  in 
the  case  of  diphtheria,  however,  the  results  would  probably  be 
better  if  more  attention  were  paid  to  the  dosage  of  the  serum. 
The  great  difficulty  is  that,  as  a  matter  of  fact,  we  have  not  the 
opportunity  of  recognising  the  presence  of  the  tetanus  bacilli 
till  they  have  begun  to  manifest  their  gravest  effects.  In 
diphtheria  we  have  a  well-marked  clinical  feature  which  draws 
attention  to  the  probable  presence  of  the  bacilli  —  a  presence 
which  can  be  readily  proved  —  and  the  curative  agent  can  thus 
be  early  applied.  In  tetanus  the  wound  in  which  the  bacilli 
exist  may  be,  as  we  have  seen,  of  the  most  trifling  character, 
and  even  when  a  well-marked  wound  exists,  the  search  for  the 
bacilli  may  be  a  matter  of  difficulty.  Still  it  might  be  well, 
when  practicable,  that  every  ragged,  unhealthy-looking  wound, 
especially  when  contaminated  with  soil,  should,  as  a  matter  of 
routine,  be  examined  bacteriologically.  In  such  cases,  un- 
doubtedly, from  time  to  time  cases  of  tetanus  would  be  detected 
early,  and  their  treatment  could  be  undertaken  with  more  hope 
of  success  than  at  present.  However,  in  the  existing  state  of 

1  The  antitetanic  serum  sent  out  by  the  Pasteur  Institute  in  Paris  has  a  strength 
of  1-1,000,000,000.  Of  this  it  is  recommended  that  50  to  100  c.c.  should  be  injected 
in  one  or  two  doses. 


392  TETANUS. 

matters,  whenever  the  first  symptoms  of  tetanus  appear,  large 
doses,  such  as  those  above  indicated,  of  a  serum  whose  strength 
is  known,  should  be  at  once  administered.  In  giving  a  prognosis 
as  to  the  probable  result,  the  two  clinical  observations  on  which, 
according  to  Behring,  chief  reliance  ought  to  be  placed,  are  the 
presence  or  absence  of  interference  with  respiration  and  the 
rapidity  with  which  the  groups  of  muscles  usually  affected  are 
attacked.  If  dyspnoea  or  irregularity  in  respiration  comes  on 
soon,  and  if  group  after  group  of  muscles  is  quickly  involved, 
then  the  outlook  is  extremely  grave.  In  addition  to  these 
points  the  duration  of  the  incubation  period  is  of  high  impor- 
tance in  forming  a  prognosis.  The  shorter  the  time  between  the 
infliction  of  a  wound  and  the  appearance  of  symptoms  the  graver 
is  the  outlook. 

The  theory  as  to  the  nature  of  antitoxic  action  will  be  dis- 
cussed later  in  the  chapter  on  Immunity. 

Methods  of  Examination  in  a  Case  of  Tetanus.  —  The  routine 
bacteriological  procedure  in  a  case  presenting  the  clinical  fea- 
tures of  tetanus  ought  to  be  as  follows :  — 

(a)  Microscopic.  —  Though  tetanus  is  not  a  disease  in  which 
the  discovery  of  the  bacilli  is  easy,  still  microscopic  examination 
should  be  undertaken  in    every  case.     From    every  wound   or 
abrasion  from  which  sufficient  discharge  can  be  obtained,  film 
preparations  ought  to   be  made,  and   stained  with  any  of   the 
ordinary  combinations,  e.g.  carbol-fuchsin  diluted  with  five  parts 
of   water.     Drumstick-shaped    spore-bearing  bacilli  are   to    be 
looked  for.     The  presence  of    such,  having   characters   corre- 
sponding to  those  of  the  tetanus  bacilli,  though  not  absolutely 
conclusive  proof  of  identification,  is  yet  sufficient  for  all  practi- 
cal  purposes.     If   only  bacilli  without  spores,  resembling  the 
tetanus   bacilli,  are  seen,  then   the  identification    can    only  be 
provisional. 

The  microscopic  examination  of  wounds  contaminated  by 
soil,  etc.,  may,  as  we  have  said,  in  some  cases  lead  to  the  antici- 
pation that  tetanus  will  probably  result. 

(b)  Cultivation.  —  The  methods  to  be  employed  in  isolating 
the   tetanus  bacilli   have  already  been  described  (p.  379).     It 
may  be  added,  however,  that  if  the  characteristic  forms  are  not 
seen    on    microscopic    examination    of    the   material   from    the 
wound,  they  may  often  be   found  by  inoculating  a  deep  tube 


MALIGNANT   (EDEMA.  393 

of  one  of  the  glucose  media  with  such  material,  and  incubating 
for  forty-eight  hours  at  37°  C.  At  the  end  of  this  period, 
spore-bearing  tetanus  bacilli  may  be  detected  microscopically, 
though  of  course  mixed  with  other  organisms. 

(c)  Inoculation.  —  Mice  and  guinea-pigs  are  the  most  suita- 
ble animals.  Inoculation  with  the  material  from  a  wound 
should  be  made  subcutaneously.  A  loopful  of  the  discharge 
introduced  at  the  root  of  the  tail  in  a  mouse  will  soon  give 
rise  to  the  characteristic  symptoms,  if  tetanus  bacilli  are  present. 

MALIGNANT  (EDEMA  (Septic/mie  de  Pasteur). 

The  organism  now  usually  known  as  the  bacillus  of  malig- 
nant oedema  is  the  same  as  that  first  discovered  by  Pasteur  and 
named  vibrion  septique.  He  described  its  characters,  distin- 
guishing it  from  the  anthrax  bacillus  which  it  somewhat  resem- 
bles morphologically,  and  also  the  lesions  produced  by  it.  He 
found  that  it  grew  only  in  anaerobic  conditions,  but  was  able 
to  cultivate  it  merely  in  an  impure  state.  It  was  more  fully 
studied  by  Koch,  who  called  it  the  bacillus  of  malignant  oedema, 
and  pointed  out  that  the  disease  produced  by  it  is  not  really  of 
the  nature  of  a  septicaemia,  as  immediately  after  death  the 
blood  is  practically  free  from  the  bacilli. 

"Malignant  oedema"  in  the  human  subject  is  usually  de- 
scribed as  a  spreading  inflammatory  oedema  attended  with  em- 
physema, and  ultimately  followed  by  gangrene  of  the  skin  and 
subjacent  parts.  In  many  cases  of  this  nature  the  bacillus  of 
malignant  oedema  is  present,  associated  with  other  organisms 
which  aid  its  spread,  whilst  in  others  it  may  be  absent.  One 
of  us  has,  however,  observed  a  case  in  which  the  bacillus  was 
present  in  pure  condition.  Here  there  occurred  intense  oedema 
with  swelling  and  induration  of  the  tissues,  and  the  formation 
of  vesicles  on  the  skin.  Those  changes  were  attended  with  a 
reddish  discoloration,  afterwards  becoming  livid.  Emphysema 
was  not  recognisable  until  the  limb  was  incised,  when  it  was 
detected,  though  in  small  degree.  Further,  the  tissues  had  a 
peculiar  heavy  but  not  putrid  odour.  The  bacillus,  which  was 
obtained  in  pure  culture,  was  present  in  enormous  numbers  in 
the  affected  tissues,  attended  by  cellular  necrosis,  serious  exu- 
dation, and  at  places  much  leucocytic  emigration.  The  picture, 


394  MALIGNANT   CEDEMA. 

in  short,  corresponded  with  that  seen  on  inoculating  a  guinea- 
pig  with  a  pure  culture.  The  term  "  malignant  oedema " 
should  be  limited  in  its  application  to  cases  in  which  the  bacil- 
lus in  question  is  present.  In  most  of  these  there  is  a  mixed 
infection ;  in  some  this  bacillus  may  be  present  alone. 

This  bacillus  has  a  very  widespread  distribution  in  nature, 
being  present  in  garden  soil,  dung,  and  various  putrefying  animal 
fluids ;  and  it  is  by  contamination  of  lacerated  wounds  by  such 
substances  that  the  disease  is  usually  set  up  in  the  human  subject. 
Malignant  oedema  can  be  readily  produced  by  inoculating  sus- 
ceptible animals,  such  as  guinea-pigs,  with  garden  soil.  The 
bacillus  is  also  often  present  in  the  intestine  of  man  and  animals, 
and  has  been  described  as  being  present  in  some  gangrenous 
conditions  originating  in  connection  with  the  intestine  in  the 
human  subject. 

Bacillus    oedematis    maligni.  —  Microscopical    Characters.  — 
The  bacillus  of  malignant  oedema  is  a  comparatively  large  or- 
ganism,   being 

•jgfr..-.  slightly       less 

than  i  /JL  in 
thickness,  that 
is,  thinner  than 
the  anthrax  ba- 
cillus. It  oc- 
curs in  the  form 
•  of  single  rods 

^fik   «<b  II    3  /*  to  10  p  in 

length,  but  both 

».""*  |£V     in    the    tissues 

and  in  cultures 
in  fluids  it  fre- 
quently grows 
out  into  long  fil- 
aments, which 
may  be  uniform 

FIG.  132.  — Film  preparation  from  the  affected  tissues  in  a  case     throughout     or 
of  malignant  oedema  in  the  human  subject,  showing  the  spore-bear-  a.-n+aA       of 

ing  bacilli.    Gentian-violet.     X  1000.  Segmented 

irregular  inter- 
vals. In  cultures  on  solid  media  it  chiefly  occurs  in  the  form  of 
shorter  rods  with  somewhat  rounded  ends.  The  rods  are  motile, 


CULTURAL   CHARACTERS.  395 

possessing  several  laterally  placed  flagella,  but  in  a  given  speci- 

men, as  a  rule,  only  a  few  bacilli  show  active  movement.     Under 

suitable  conditions  they  form  spores  which  are  usually  near  the 

centre  of   the  rods  and   have 

an  oval  shape,  their  thickness  i  *jy^ 

somewhat  exceeding  that  of  the  * 

bacillus  (Figs.  132,  133).     The  '  J  '    r  ^          r      I 

bacillus  can  be  readily  stained      :\       /»*      -  *         \  CC\*    *- 

by    any   of    the    basic    aniline     •»  ^     **f~    t& 

stains,  but  loses  the  colour  in 

Gram's    method,    in    this    way       ^  ^   *"*>/*•     A 

differing     from     the     anthrax      I       ^  *  /"*  ^        i      V^* 

bacillus. 


Characters    of    Cultures.  — 


% 
V 


'  «••  n, 

This  organism  grows  readily  at  •*?  1  --'-' 

ordinary  temperature,  but  only       FlG  I33._Bacil]us  of  malignant 

Under  anaerobic  Conditions.      In    showing  spores.     From  a  culture  in  glucose 

a    nunrture    culture    in    a    rWn    agar'  incubated  for  three  days  at  37°  C. 
1    a    Qeep    Stained  with  weak  Carbol-fuchsin.     x  1000. 

tube    of   glucose    gelatin,    the 

growth  appears  as  a  whitish  line  giving  off  minute  short  processes, 
the  growth,  of  course,  not  reaching  the  surface  of  the  medium. 
Soon  liquefaction  occurs,  and  a  long  fluid  funnel  is  formed,  with 
turbid  contents  and  flocculent  masses  of  growth  at  the  bottom. 
At  the  same  time  bubbles  of  gas  are  given  off,  which  may  split 
up  the  gelatin.  The  colonies  in  gelatin  plates  under  anaerobic 
conditions  appear  first  as  small  whitish  points  which  under 
the  microscope  show  a  radiating  appearance  at  the  periphery, 
resembling  the  colonies  of  the  bacillus  subtilis.  Soon,  however, 
liquefaction  occurs  around  the  colonies,  and  spheres  with  turbid 
contents  result  ;  gas  is  developed  around  the  colonies. 

In  deep  tubes  of  glucose  agar  at  37°  C.,  growth  is  extremely 
rapid.  Along  the  line  of  puncture,  growth  appears  as  a  some- 
what broad  white  line  with  short  lateral  projections  here  and 
there  (Fig.  134,  B).  Here  also  gas  may  be  formed,  but  this  is 
most  marked  in  a  shake-culture,  in  which  the  medium  becomes 
cracked  in  various  directions,  and  may  be  pushed  upwards  so 
high  as  to  displace  the  cotton-wool  plug.  The  cultures  possess 
a  peculiar  heavy,  though  not  putrid,  odour.  In  litmus  milk  a 
feeble  acidity  is  produced  with  an  occasional  flocculent  precipi- 
tation of  casein  at  the  end  of  forty-eight  hours  ;  this  is  soon 


396 


MALIGNANT   OEDEMA. 


followed  by  a  peptonising  process  which   advances  so  that  the 
whole  of  the  medium  becomes  converted  into  a  muddy-coloured 

whey  that  gives  off  a  heavy 
putrid  odour. 

Spore  formation  occurs 
above  20°  C.,  and  is  usu- 
ally well  seen  within  forty- 
eight  hours  at  37°  C.  The 
spores  have  the  usual  high 
powers  of  resistance,  and 
may  be  kept  for  months  in 
the  dried  condition  without 
being  killed. 

Experimental  Inocu- 
lation. —  A  considerable 
number  of  animals  —  the 

W  guinea-pig,    rabbit,    sheep, 

and  goat,  for  example  — 
I  are  susceptible  to  inocula- 
I  tion  with  this  organism. 
•  The  ox  is  said  to  be  quite 
immune  to  experimental 
inoculation,  though  it  can, 
under  certain  conditions, 
contract  the  disease  by 
natural  channels.  The 
guinea-pig  is  the  animal  most  convenient  for  experimental 
inoculation.  When  the  disease  is  set  up  in  the  guinea-pig 
by  subcutaneous  inoculation  with  garden  soil,  death  usually 
occurs  in  about  twenty-four  to  forty-eight  hours.  There  is  an 
intense  inflammatory  oedema  around  the  site  of  inoculation, 
which  extends  over  the  wall  of  the  abdomen  and  thorax.  The 
skin  and  subcutaneous  tissue  are  infiltrated  with  a  reddish- 
brown  fluid  and  softened ;  they  contain  bubbles  of  gas  and  are 
at  places  gangrenous.  The  superficial  muscles  are  also  in- 
volved. These  parts  have  a  very  putrid  odour.  The  internal 
organs  are  congested,  the  spleen  soft  but  not  much  enlarged. 
In  such  conditions  the  bacillus  of  malignant  oedema,  both  in 
short  and  long  forms,  will  be  found  in  the  affected  tissues  along 
with  various  other  organisms.  Spores  may  be  present,  espe- 


FlG.  134.  —  Stab-cultures  in  agar,  five  days'  growth 
at  37°  C.     Natural  size. 

A.    Tetanus  bacillus.      B.    Bacillus  of  malignant  oedema. 
C.    Bacillus  of  quarter-evil  (Rauschbrand). 


IMMUNITY:  DIAGNOSIS.  397 

cially  when  the  examination  is  made  some  time  after  the  death 
of  the  animal.  If  the  animal  is  examined  immediately  after 
death,  a  few  of  the  bacilli  may  be  present  in  the  peritoneum  and 
pleurae,  usually  in  the  form  of  long  motile  filaments,  but  they 
are  almost  invariably  absent  from  the  blood.  A  short  time  after 
death,  however,  they  spread  directly  into  the  blood  and  various 
organs,  and  may  then  be  found  in  considerable  numbers. 

Subcutaneous  inoculation  with  pure  cultures  of  the  bacillus 
of  malignant  oedema  produces  chiefly  a  spreading  bloody 
oedema,  the  muscles  being  softened  and  partly  necrosed  ;  but 
there  is  little  formation  of  gas,  and  the  putrid  odour  is  almost 
absent. 

When  the  bacilli  are  injected  into  mice,  however,  they  enter 
and  multiply  in  the  blood  stream,  and  they  are  found  in  con- 
siderable numbers  in  the  various  organs,  so  that  a  condition 
not  unlike  that  of  anthrax  is  found.  The  spleen  also  is  much 
swollen. 

The  virulence  of  the  bacillus  of  malignant  oedema  varies  con- 
siderably in  different  cases,  and  it  always  becomes  diminished  in 
cultures  grown  for  some  time.  To  produce  a  fatal  disease,  a 
relatively  large  number  of  the  organisms  is  necessary,  and  these 
must  be  introduced  deeply  into  the  tissues,  inoculation  by  scari- 
fication being  followed  by  no  result.  A  smaller  dose  produces 
a  fatal  result  when  injected  along  with  various  other  organisms 
(bacillus  prodigiosus,  etc.). 

Immunity.  —  Malignant  oedema  was  one  of  the  first  diseases 
against  which  immunity  was  produced  by  injection  of  toxins. 
The  filtered  cultures  of  the  bacillus  in  sufficient  doses  produce 
death  with  the  same  symptoms  as  those  caused  by  the  living 
organisms,  but  a  relatively  large  quantity  is  necessary.  Cham- 
berland  and  Roux(i88/)  found  that  if  guinea-pigs  were  injected 
with  several  non-fatal  doses  of  cultures  sterilised  by  heat  or 
freed  from  the  bacilli  by  filtration,  immunity  against  the  living 
organism  could  be  developed  in  a  comparatively  short  time. 
They  found  that  the  filtered  serum  of  animals  dead  of  the  dis- 
ease is  more  highly  toxic,  and  also  gives  immunity  when 
injected  in  small  doses.  These  experiments  have  been  con- 
firmed by  Sanfelice. 

Methods  of  Diagnosis.  —  In  a  case  of  supposed  malignant 
oedema,  the  fluid  from  the  affected  tissues  ought  first  to  be 


398  BACILLUS    BOTULINUS. 

examined  microscopically  to  ascertain  the  characters  of  the 
organisms  present.  Though  it  is  not  possible  to  identify  ab- 
solutely the  bacillus  of  malignant  oedema  without  cultivating  it, 
the  presence  of  spore-bearing  bacilli  with  the  characters  de- 
scribed above  is  highly  suspicious  (Fig.  132).  In  such  a  case 
the  fluid  containing  the  bacilli  should  be  first  exposed  to  a 
temperature  of  80°  C.  for  half  an  hour,  and  then  a  deep  glucose 
agar  tube  should  be  inoculated.  In  this  way  the  spore-free 
organisms  are  killed  off.  Pure  cultures  may  be  thus  obtained, 
or  this  procedure  may  require  to  be  followed  by  the  roll-tube 
method  under  anaerobic  conditions.  An  inoculation  experi- 
ment, if  available,  may  also  be  made  on  a  guinea-pig. 

BACILLUS  BOTULINUS. 

The  term  "  meat-poisoning  "  embraces  a  number  of  conditions 
produced  by  different  agents,  and  the  relation  of  the  bacillus  of 
Gaertner  to  one  class  of  case  has  already  been  discussed.  Another 
group  was  shown  by  Van  Ermengem  in  1896  to  be  caused  by  an 
anaerobic  bacillus  to  which  he  gave  the  name  bacillus  botulinus. 
He  cultivated  the  organism  from  a  sample  of  ham,  the  ingestion 
of  which  in  the  raw  condition  had  produced  a  number  of  cases 
of  poisoning,  some  of  them  followed  by  fatal  result.  The 
symptoms  in  these  cases  closely  corresponded  with  those  occur- 
ring in  the  so-called  "  sausage-poisoning "  met  with  from  time 
to  time  in  Germany  and  other  countries  where  sausages  and 
ham  are  eaten  in  an  imperfectly  cooked  condition.  Such  cases 
form  a  fairly  well-defined  group,  the  symptoms  in  which  are 
chiefly  referable  to  an  action  on  the  medulla,  and,  as  will  be 
detailed  below,  similar  symptoms  have  been  experimentally 
produced  by  means  of  the  bacillus  mentioned  or  its  toxins. 
The  chief  symptoms  of  this  variety  of  botulismus,  as  detailed 
by  Van  Ermengem,  are  disordered  secretion  in  the  mouth  and 
nose,  more  or  less  marked  ophthalmoplegia,  externa  and  interna 
(dilated  pupil,  ptosis,  etc.),  dysphagia,  and  sometimes  aphagia 
with  aphonia,  marked  constipation  and  retention  of  urine,  and 
in  fatal  cases  interference  with  the  cardiac  and  respiratory 
centres.  Along  with  these  there  is  practically  no  fever  and  no 
interference  with  the  intellectual  faculties.  The  symptoms 
commence  at  earliest  twelve  to  twenty-four  hours  after  inges- 


MICROSCOPIC   AND   CULTURAL   CHARACTERS.  399 

tion  of  the  poison.  From  the  ham  in  question,  which  was  not 
decomposed  in  the  ordinary  sense,  Van  Ermengem  obtained 
numerous  colonies  of  this  bacillus,  the  leading  characters  of 
which  are  given  below.  It  may  be  added  that  Romer  obtained 
practically  the  same  results  as  Van  Ermengem  in  a  similar  con- 
dition, and  that  the  bacillus  botulinus  has  been  cultivated  by 
Kemper  from  the  intestine  of  the  pig. 

Microscopical  and  Cultural  Characters.  —  The  organism  is  a 
bacillus  of  considerable  size,  measuring  4  to  9  ft  in  length  and 
.9  to  1.2  p  in  thickness;  it  has  somewhat  rounded  ends  and  some- 
times is  seen  in  a  spindle  form.  It  is  often  arranged  in  pairs, 
sometimes  in  short  threads.  Under  certain  conditions  it  forms 
spores  which  are  oval  in  shape,  usually  terminal  in  position,  and 
a  little  thicker  than  the  bacilli.  It  is  a  motile  organism  and  has 
4  to  8  lateral  flagella  of  wavy  form.  It  stains  readily  with  the 
ordinary  dyes,  and  also  retains  the  colour  in  Gram's  method, 
though  care  must  be  employed  in  decolorising. 

The  organism  can  be  readily  cultivated  on  the  ordinary  media, 
but  only  under  strictly  anaerobic  conditions.  In  glucose  gelatin 
a  whitish  line  of  growth  forms  with  lateral  offshoots,  but  lique- 
faction with  abundant  gas  formation  soon  occurs.  In  gelatin 
plates  the  colonies  after  four  to  six  days  are  somewhat  charac- 
teristic ;  they  appear  to  the  naked  eye  as  small  semi-transparent 
spheres,  and  these  on  examination  under  a  low  power  of  the 
microscope  have  a  yellowish-brown  colour  and  are  seen  to  be 
composed  of  granules  which  show  a  streaming  movement, 
especially  at  the  periphery.  Cultures  in  glucose  agar  resemble 
those  of  certain  other  anaerobes  ;  there  is  abundant  development 
of  gas,  and  the  medium  is  split  up  in  various  directions.  The 
cultures  have  a  -rancid,  though  not  foul,  odour,  due  chiefly  to 
the  development  of  butyric  acid.  The  optimum  temperature  is 
below  that  of  the  body,  viz.,  between  20°  and  30°  C. ;  at  the 
body  temperature  growth  is  slower  and  less  abundant  and  spore 
formation  does  not  occur. 

Pathogenic  Effects.  —  Like  the  tetanus  bacillus,  the  bacillus 
botulinus  has  little  power  of  flourishing  in  the  tissues,  whereas 
it  produces  a  very  powerful  toxin.  Van  Ermengem  found  that 
the  characteristic  symptoms  could  be  produced  in  certain  ani- 
mals by  administering^ watery  extracts  of  the  infected  ham  or 
cultures  either  by  the  alimentary  canal  or  by  subcutaneous 


400  BACILLUS    BOTULINUS. 

injection.  Here  also  there  is  a  period  of  incubation  of  not  less 
than  six  to  twelve  hours  before  the  symptoms  appear,  and  when 
the  dose  is  small  a  somewhat  chronic  condition  may  result  in 
which  local  paralyses  form  a  striking  feature.  The  character- 
istic effects  can  also  be  produced  by  means  of  the  filtered  toxin 
by  either  of  the  methods  mentioned,  though  in  the  case  of 
administration  by  the  alimentary  canal  the  dose  requires  to  be 
larger.  Here  also,  as  in  the  case  of  the  tetanus  poison,  the 
potency  of  the  toxin  is  remarkable,  the  fatal  dose  for  a  guinea- 
pig  of  250  grammes'  weight  being  in  some  instances  .0005  c.c. 
of  the  filtered  toxin.  In  cases  of  poisoning  in  the  human  sub- 
ject the  effects  would  accordingly  appear  to  be  produced  by 
absorption  of  the  toxin  from  the  alimentary  canal;  it  is  only 
after  or  immediately  before  death  that  a  few  bacilli  may  enter 
the  tissues.  Van  Ermengem  obtained  a  few  colonies  from  the 
spleen  of  a  patient  who  had  died  from  ham-poisoning.  The 
properties  of  the  botulinus  toxin  have  been  investigated  and 
have  been  found  to  correspond  closely,  as  regards  relative  insta- 
bility, conditions  of  precipitation,  etc.,  with  the  toxins  of  diph- 
theria and  tetanus.  An  antitoxin  has  also  been  prepared  by 
Kempner  by  the  usual  methods,  and  has  been  shown  not  only 
to  have  the  neutralising  property,  but  to  have  considerable 
therapeutical  value  when  administered  some  hours  after  the  toxin. 
The  direct  combining  affinity  of  the  toxin  for  the  central  nervous 
has  been  demonstrated  by  Kempner  and  Schepilewsky  by  the 
same  methods  as  Wassermann  and  Takaki  employed  in  the 
case  of  the  tetanus  toxin.  The  condition  of  the  nerve  cells  in 
experimental  poisoning  with  the  botulinus  toxin  has  been  in- 
vestigated independently  by  Marinesco  and  by  Kempner  and 
Pollack,  and  these  observers  agree  as  to  the  occurrence  of 
marked  degenerative  changes,  especially  in  the  motor  cells 
in  the  spinal  cord  and  medulla.  Marinesco  also  observed 
hypertrophy  and  proliferation  of  the  neuroglia  cells  around 
them. 

These  observations,  therefore,  show  that  in  one  variety  of 
meat-poisoning  the  symptoms  are  produced  by  the  absorption 
of  the  toxins  of  the  bacillus  botulinus  from  the  alimentary  canal, 
and,  as  Van  Ermengem  points  out,  it  is  of  special  importance  to 
note  that  the  meat  may  be  extensively  contaminated  with  this 
bacillus  and  may  contain  relatively  large  quantities  of  its  toxins 


QUARTER-EVIL.  401 

without  the  ordinary  signs  of  decomposition  being  present.  The 
production  of  an  extracellular  toxin  by  this  organism,  with  ex- 
tremely potent  action  on  the  nervous  system,  is  a  fact  of  great 
scientific  interest,  and  has  a  bearing  on  the  etiology  of  other 
obscure  nervous  affections. 

QUARTER-EVIL  (GERMAN,  RAUSCHBRAND  ;  FRENCH,  CHARBON 
SYMPTOMATIQUE)  . 

The  characters  of  the  bacillus  need  be  only  briefly  described,  as,  so  far  as 
is  known,  it  never  infects  the  human  subject.  The  natural  disease,  which 
specially  occurs  in  certain  localities,  affects  chiefly  sheep,  cattle,  and  goats. 
Infection  takes  place  by  some  wound  of  the  surface,  and  there  spreads  in  the 
region  around,  inflammatory  swelling  attended  by  bloody  oedema  and  em- 
physema of  the  tissues.  The  part  becomes  greatly  swollen,  and  of  a  dark, 
almost  black,  colour.  Hence  the  name  blackleg  by  which  the  disease  is  some- 
times known.  The  bacillus  which  produces  this  condition  is  present  in  large 
numbers  in  the  affected  tissues,  associated  with  other  organisms,  and  also 
occurs  in  small  numbers  in  the  blood  of  internal  organs. 

Bacillus  anthracis  symptomatici.  —  The  bacillus  morphologically  closely 
resembles  that  of  malignant  oedema.  Like  the  latter,  also,  it  is  a  strict  ana- 
erobe, and  its  conditions  of  growth  as  regards  temperature  are  also  similar. 
It  is,  however,  somewhat  thicker,  and  .  - 

does  not  usually  form  such  long  fila-  ^        /  ^^  - 

ments.     Moreover  the  spores,  which  .  ^  ~  \ 

are  of  oval  shape  and  broader  than  I  "^     ^ 

the  bacillus,  are  almost  invariably  situ-  \^^      %jr* 

ated  close  to  one  extremity,  though  X      S  ^ 

not  actually  terminal  (Fig.  135).    The  •»  "  V   V.   ^  \ 

characters  of  the  cultures,  also,  resem-     A 
ble  those  of  the  bacillus  of  malignant 

oedema,  but  in  a  stab-culture  in  glu-  t         ^  <r~     y«* 

cose  agar  there  are  more  numerous  and  Jt  ^* ' 

longer  lateral   offshoots,  the  growth  H         "•** 

being  also  more  luxuriant  (Fig.  1 34,  C). 
This  bacillus  is  actively  motile,  and 

possesses  numerous  lateral  flagella.  FIG.  135.  —  Bacillus  of  quarter-evil,  show- 
It  does  not  retain  the  Stain  by  Gram's  ing  spores.  From  a  culture  in  glucose  agar, 
method  incubated  for  three  days  at  37°  C.  Stained 

with  weak  carbol-fuchsin.     X  icoo. 

The  disease  can  be  readily  pro- 
duced in  various  animals,  e.g.  guinea-pigs,  by  inoculation  with  the  affected 
tissues  of  diseased  animals,  and  also  by  means  of  pure  cultures,  though  an 
intramuscular  injection  of  a  considerable  amount  of  the  latter  is  sometimes 
necessary.  The  condition  produced  in  this  way  closely  resembles  that  in 
malignant  oedema,  though  there  is  said  to  be  more  formation  of  gas  in  the 
tissues.  Rabbits  are  very  immune  against  this  disease,  whilst  they  are  com- 
paratively susceptible  to  malignant  oedema.  As  in  the  case  of  tetanus,  inocu- 


402  BACILLUS   AEROGENES   CAPSULATUS. 

lation  with  living  spores  which  have  been  deprived  of  adherent  toxin  by  heat 
does  not  produce  the  disease. 

The  disease  is  one  against  which  immunity  can  be  readily  produced  in 
various  ways,  and  methods  of  preventive  inoculation  have  been  adopted  in  the 
case  of  animals  liable  to  suffer  from  it.  This  subject  was  specially  worked  out 
by  Arloing,  Cornevin,  and  Thomas,  and  later  by  others.  Immunity  may  be 
produced  by  injection  with  a  non-fatal  dose  of  the  virus  (i.e.  the  oadematous 
fluid  found  in  the  tissues  of  affected  animals  and  which  contains  the  bacilli), 
or  by  injection  with  larger  quantities  of  the  virus  attenuated  by  heat,  drying, 
etc.  It  can  be  produced  also  by  the  products  of  the  bacilli  obtained  by  filtra- 
tion of  cultures. 

BACILLUS  AEROGENES  CAPSULATUS. 

Bacillus  aerogenes  capsulatus  (Welch).  —  Synonym  :  —  Bacil- 
lus Welchii  (Migula). 

Introductory.  —  This  organism  was  discovered  by  Welch  in 
1891  in  the  blood  and  tissues  of  a  tuberculous  person  who  died 
from  rupture  of  an  aortic  aneurism.  The  skin  over  the  body 
was  markedly  emphysematous,  gas  was  found  in  the  heart  and 
great  vessels,  in  the  loose  tissues  of  the  abdominal  wall,  and  in 
the  various  solid  viscera,  which  presented  the  picture  called  by 
the  Germans,  " Schaumorgane  "  A  full  description  of  the  case 
and  of  the  bacillus  was  published  by  Welch  and  Nuttall  the 
following  year.  In  1893  E.  Fraenkel  published  a  monograph 
on  emphysematous  gangrene  and  therein  described  an  anaero- 
bic bacillus  as  being  the  probable  cause,  naming  it  B.  phleg- 
mones  emphysematosae,  which,  in  all  its  details,  morphological 
and  cultural,  corresponded  to  that  described  previously  by  Welch 
and  Nuttall,  and  later  acknowledged  by  Fraenkel  as  identical. 
Since  then  this  organism  has  been  observed  over  and  over  again 
by  European  workers,  mainly  ignorant  of  the  American  work 
upon  it,  and  given  such  names  as  B.  perfringens,  B.  enteritidis 
sporogenes,  Granulo-bacillus  immobilis,  etc.  In  America  in  re- 
cent years  papers  have  appeared  by  Welch  and  Flexner,  How- 
ard, Bloodgood,  Fulton  and  Pratt,  and  many  others,  recording 
the  occurrence  of  B.  aerogenes  capsulatus  in  the  human  body 
under  varied  conditions.  Welch,  in  1900,  reviewed  the  whole 
literature  and  recorded  many  hitherto  unpublished  facts. 

Habitat.  —  The  bacillus  is  widely  distributed,  being  found 
in  the  intestinal  contents  of  most  mammals,  in  sewage,  in  river 
water,  in  garden  and  field  earth,  in  dust,  and  in  milk  and  many 
raw  foods. 


MORPHOLOGY  AND   PATHOGENESIS.  403 

Morphology.  —  The  bacillus  resembles  somewhat  B.  anthracis, 
but  generally  is  stouter  and  more  variable  in  length  (Fig.  136), 
on  an  average  3-6  /JL,  but  it  may  be  found  in  pairs  so  short  as  to 
resemble  diplococci,  or  so  long  as  to  form  filaments,  and  chains 
of  4  to  30  units  may  be  met  with.  The  ends  of  free  bacilli  are 
obtusely  rounded,  whilst  those  of  organisms  in  a  chain  are  square 
cut.  The  bacillus  is  usually  possessed  of  a  capsule,  especially 
when  found  in  body  fluids,  rarely 
in  artificial  media,  but  at  all  times 
the  capsule  stains  with  difficulty. 

Spores  are  generally  to  be 
found  in  blood-serum  cultures 
after  four  days'  incubation,  and 
occasionally  they  have  been  ob- 
served in  cultures  in  all  of  the 
common  media.  It  would  ap- 
pear, however,  that  some  races 
are  devoid  of  the  power  to  form 

Spores.         The     majority     of     the  FIG.  136.  —  Bacillus  aerogenes  capsu- 

spores  are  oval  in  form,  less  fre-    'atus'  "^?how!ng  one  larf  •  /ncapsuied 

form.    The  others  in  the  field  are  of  the 
quently  round,  and    may  be   Situ-     same  species,  but  smaller.     (Dr.  Charles 

ated  in  the  cell  either  centrally,  H"  Potten)  x  Iooa 
towards  one  end,  or  at  the  end,  and  are  usually  of  slightly  greater 
diameter  than  the  bacillus  itself.  Two  have  never  been  found 
in  one  cell.  They  stain  readily  by  any  of  the  ordinary  spore- 
staining  methods.  They  withstand  the  temperature  of  boiling 
water  for  six  minutes,  but  are  killed  by  eight  minutes'  boiling. 
The  vegetative  cells,  however,  perish  by  an  exposure  of  ten 
minutes  in  moist  heat  at  58°  C.  The  bacilli  are  not  possessed 
of  flagella  and  are  non-motile.  They  stain  by  Gram's  method. 
Pathogenesis.  —  In  man,  as  pointed  out  by  Fraenkel,  Welch, 
Bloodgood,  and  others,  this  bacillus  is  the  most  frequent  cause 
of  emphysematous  gangrene.  It  has  been  repeatedly  found  in 
the  inflammatory  subcutaneous  emphysema  following  upon  in- 
jections of  salt  solution,  due  to  imperfect  sterilisation  of  the 
instrument  or  fluid.  It  has  been  cultivated  from  the  blood  dur- 
ing life  in  two  cases  at  least  —  by  Gwyn  in  a  case  of  acute  cho- 
rea which  ended  fatally,  and  by  Cole,  recently,  in  the  Johns 
Hopkins  Hospital,  from  the  blood  of  a  man  whose  leg  had  been 
crushed  in  a  railroad  accident  and  was  at  the  time '  of  culture 


404  BACILLUS   AEROGENES   CAPSULATUS. 

markedly  emphysematous ;  it  is  notable  that  in  these  cases  no 
gas  was  found  in  the  blood  during  abstraction  of  it  with  the 
syringe,  although  closely  watched  for. 

In  the  lower  animals,  infections  by  this  bacillus  have  been 
rarely  met  with  under  natural  conditions.  Harris,  however,  has 
observed  local  abscesses  in  the  dog  and  rabbit  following  upon 
simple  surgical  procedures.  Under  artificial  conditions  B.  aerog- 
enes  capsulatus  is  pathogenic  for  the  guinea-pig  and  pigeon, 
but  practically  not  for  the  rabbit  and  mouse.  Experience  has 
shown  that  the  virulence  of  the  bacillus  towards  guinea-pigs, 
when  injected  subcutaneously,  varies  exceedingly.  Certain  races 
are  absolutely  harmless ;  some  produce  local  swelling,  with  or 
without  necrosis  and  ulceration,  whilst  others  will  produce  death, 
accompanied  by  subcutaneous  emphysema  and  a  peculiar  diges- 
tive-like action  upon  the  subcutaneous  tissues  and  abdominal 
muscles,  so  that  the  intestines  can  plainly  be  seen  lying  in  the 
cavity.  There  is  also  much  bloody  serous  fluid  along  with  this 
necrotic  process. 

Cultural  Characters.  —  The  bacillus  is  an  obligate  anaerobe. 
The  surface  colonies  on  plain-agar  plates  appear  to  the  unaided 
eyes  to  be  well  circumscribed,  round,  flat,  grey-white,  translu- 
cent, smooth,  and  glossy,  with  a  diameter  varying  from  1.5  to  4 
mm.  The  deep  colonies  are  seen  as  small  white  points,  some- 
times indistinctly  limited.  Under  the  low  power  of  the  microscope 
the  surface  colonies  are  found  to  be  not  infrequently  irregular, 
with  a  central  mass  or  nucleus  embedded  in  a  surrounding  finely 
granular  growth  of  a  light  brown-yellow  colour,  whose  periphery 
is  regular  and  often  delicately  fringed.  The  deep  colonies  are 
usually  of  a  clear-cut  oval  or  round  shape,  but  occasionally  they 
appear  to  be  surrounded  with  woolly  outgrowths,  similar  in 
character  to  those  of  B.  tetani.  On  agar  slants  the  surface 
growth  is  thin,  grey,  translucent,  glossy,  and  veil-like;  at  times 
difficult  to  detect. 

In  shake-cultures,  especially  in  glucose  agar  or  ghtcose  gelatin, 
gas  formation  is  very  profuse,  so  that  the  medium  becomes  split 
up  into  layers,  the  uppermost  often  pressing  against  the  cotton 
plug.  There  is  an  accompanying  heavy  peculiar  odour  lacking 
putrefactive  taint.  Upon  potato  the  growth  is  usually  scant, 
invisible,  or  quite  negative.  Occasionally,  however,  it  is  well 
marked,  of  a  yellow-white  colour,  semi-lustrous,  moist,  and  show- 


CULTURAL   CHARACTERS. 


405 


ing  embedded  gas  bubbles.  It  is  the  rule  to  find  gas  bubbles 
lying  in  the  fluid  between  the  potato  and  the  walls  of  the  tube. 
The  medium  never  becomes  discoloured.  Bouillon  is  heavily 
clouded,  and  occasionally  gas  bubbles  are  seen  on  top.  The 
precipitated  growth  is  semi-flocculent,  white,  and  diffuses  on 
agitation.  In  litmus  milk  there  occurs  the 
most  characteristic  reaction.  At  the  end  of 
twenty-four  hours  the  reaction  is  strongly' 
acid,  the  casein  is  found  to  be  coagulated 
firmly,  but  at  the  same  time  is  more  or  less 
completely  riddled  by  cavities  caused  by  gas 
formation  (Fig.  137).  The  resulting  whey  is 
usually  quite  clear;  occasionally  it  may  be 
turbid  and  yellow.  Viscidity  is  present  on 
rare  occasions.  Now  and  again  a  layer  of 
gas  bubbles  may  cover  the  surface  of  the  fluid. 
There  is  a  strong  odour  of  butyric  acid  pres- 
ent. Gelatin  may  be  slowly  liquefied  by 
some  races  of  the  bacillus,  or  may  merely 
undergo  a  softening  without  any  definite 
liquefying ;  this  latter  seems  to  be  more 
common.  Gas  formation  may  occur,  due 
to  presence  of  muscle  sugar  in  the  meat 
extract.  On  solidified  blood  scrum  along  the 
line  of  inoculation  is  found  a  rich  cream- 
coloured,  elevated,  glossy  growth,  which 
at  the  end  of  a  week  seems  to  exercise  a 
feeble  peptonising  power,  denoted  by  a 
small  furrow  forming  in  the  medium  upon 
which  the  growth  rests,  but  this  never  goes  on  to  liquefaction. 

Lactose,  saccharose,  and  mannite  are  actively  fermented, 
with  accompanying  gas  formation ;  so  is  dextrose-free  bouillon 
and  ascitic  fluid,  and  from  these  facts  Welch  is  of  the  opinion 
that  the  bacillus  is  capable  of  forming  gas  from  proteid  material 
alone. 

Significance.  —  Its  presence  in  the  tissues  during  life  is  to  be 
looked  upon  as  serious  in  most  cases  and  deserving  of  prompt 
attention,  surgical  or  otherwise.  Post  mortem,  in  the  absence  of 
any  possible  external  point  of  entry,  its  existence  is  a  matter  of 
no  importance,  as,  like  the  colon  bacillus,  it  is  capable  of  wan- 


FlG.  137.  —  B.  aerog- 
enes  capsulatus.  A  24- 
hour-old  milk  culture, 
showing  typical  coagula- 
tion of  the  casein.  Natu- 
ral size. 


406  BACILLUS   AEROGENES   CAPSULATUS. 

dering  into  the  circulation  from  the  intestine  just  before,  or 
immediately  after,  death. 

Isolation  and  Identification.  —  The  suspected  material  should 
be  transferred  in  varying  quantities  to  milk  and  incubated  under 
anaerobic  conditions  for  forty-eight  to  seventy-two  hours,  when 
the  typical  reaction  should  be  manifest.  An  incomplete  reac- 
tion may  be  brought  about  by  the  partial  overgrowth  of  other 
bacteria,  but  usually  upon  sub-culture  in  the  same  medium  it 
will  obtain  a  sufficient  start  to  bring  about  the  typical  change. 
To  finally  obtain  the  bacillus  in  purity,  and  at  the  same  time  to 
have  a  positive  means  of  identification,  Professor  Welch  has 
devised  a  very  ingenious  method :  From  |-i  c.c.  of  such  a  milk 
culture,  or  the  same  quantity  of  a  suspension  made  from  a  solid 
medium,  is  injected  into  the  ear  vein  of  a  rabbit,  and  to  insure  a 
thorough  distribution  of  the  organisms  three  minutes  are  allowed 
to  pass  before  the  next  step  is  taken,  which  is  the  killing  of  the 
animal  by  a  sharp  blow  on  the  back  of  the  head.  The  body  is 
now  placed  in  the  thermostat  for  seven  or  eight  hours,  or  left  at 
room  temperature  for  eighteen  or  twenty-four  hours,  and  at  the 
end  of  either  of  those  periods  the  animal  will  usually  be  found 
to  be  greatly  bloated,  due  to  accumulation  of  gas  in  the  subcu- 
taneous tissue  and  in  the  body  cavities.  As  the  gas  is  highly 
inflammable  a  lighted  match  brought  to  a  puncture  in  the  ab- 
dominal wall,  or  elsewhere,  will  produce  a  bright  blue  flame 
when  in  contact  with  the  escaping  gas.  The  organs  of  the 
animal  will  be  found  to  be  full  of  gas  and  more  or  less  broken 
down.  The  organism  is  then  found  in  enormous  numbers  every- 
where and  readily  identified  by  its  capsule,  absence  of  motility, 
staining  by  Gram,  and  later,  if  some  serum  from  the  animal  is 
sealed  up  in  a  small  tube  and  placed  in  the  thermostat  it  will  be 
found  that  many  of  the  bacilli  have  formed  spores.  This  latter 
fact  was  pointed  out  by  E.  Klein  in  his  studies  on  his  B.  enteri- 
tidis  sporogenes  (B.  aerogenes  capsulatus). 


CHAPTER   XVIII. 

CHOLERA. 

Introductory.  —  It  is  no  exaggeration  of  the  facts  to  say  that 
previously  to  1883  practically  nothing  of  value  was  known  re- 
garding the  nature  of  the  virus  of  cholera.  In  that  year  Koch 
was  sent  to  Egypt,  where  the  disease  had  broken  out,  in  charge 
of  a  commission  for  the  purpose  of  investigating  its  nature. 
In  the  course  of  his  researches  he  discovered  the  organism 
now  generally  known  as  the  comma  bacillus  or  the  cholera 
spirillum.  Further  observations  carried  out  in  nearly  a  hundred 
cases,  chiefly  in  India,  convinced  him  of  the  constant  presence 
of  this  organism  in  cholera  and  of  its  causal  relationship  to  the 
disease.  He  also  obtained  pure  cultures  of  the  organism  from  a 
large  number  of  cases  of  cholera,  and  described  their  characters. 
The  results  of  his  researches  were  given  at  the  first  Cholera 
Conference  at  Berlin  in  1884.  The  general  conclusions  at 
which  Koch  arrived  received,  in  the  main,  confirmation  from 
the  investigations  of  others,  though  some  criticism  arose,  espe- 
cially as  regards  the  uniformity  of  the  characters  of  the  cholera 
spirillum. 

Since  Koch's  discovery,  and  especially  during  the  epidemic 
in  Europe  in  1892-93,  spirilla  have  been  cultivated  from  cases 
of  cholera  in  a  great  many  different  localities,  and  though  this 
extensive  investigation  has  revealed  the  invariable  presence  in 
•true  cholera  of  organisms  resembling  more  or  less  closely  Koch's 
spirillum,  certain  difficulties  have  arisen.  For  it  has  been  found 
that  the  cultures  obtained  from  different  places  have  shown 
considerable  variations  in  their  characters,  and,  further,  spirilla 
which  closely  resemble  Koch's  cholera  spirillum  have  been  cul- 
tivated from  sources  other  than  cases  of  true  cholera.  There 
has  therefore  been  much  controversy,  on  the  one  hand,  as  to  the 
signification  of  these  variations  —  whether  they  constitute  differ- 
ent species,  or  whether  they  are  to  be  regarded  as  indicating 

407 


408  CHOLERA. 

distinct  species  or  merely  varieties  of  the  same  species  —  and 
on  the  other  hand,  as  to  the  means  of  distinguishing  the  cholera 
spirillum  from  other  species  which  resemble  it.  These  questions 
will  be  discussed  below. 

In  considering  the  bacteriology  of  cholera,  it  is  to  be  borne  in 
mind  that  in  this  disease,  in  addition  to  the  evidence  of  great 
intestinal  irritation,  accompanied  by  profuse  watery  discharge, 
and  often  by  vomiting,  there  are  also  symptoms  of  general  sys- 
temic disturbance  which  cannot  be  accounted  for  merely  by 
the  withdrawal  of  water  and  certain  substances  from  the  system. 
Such  symptoms  include  the  profound  general  prostration,  cramps 
in  the  muscles,  extreme  cardiac  depression,  the  cold  and  clammy 
condition  of  the  surface,  the  subnormal  temperature,  suppression 
of  urine,  etc.  These  taken  in  their  entirety  are  indications  of  a 
general  poisoning  in  which  the  circulatory  and  thermo-regulatory 
mechanisms  are  specially  involved.  In  some,  though  rare,  cases 
known  as  cJiolcra  sicca,  general  collapse  occurs  with  remarkable 
suddenness,  and  is  rapidly  followed  by  a  fatal  result,  whilst  there 
is  little  or  no  evacuation  from  the  bowel,  though  post  mortem  the 
intestine  is  distended  with  fluid  contents.  As  the  characteristic 
organisms  in  cholera  are  found  only  in  the  intestine,  the  general 
disturbances  are  to  be  regarded  as  the  result  of  toxic  substances 
absorbed  from  the  bowel.  It  is  also  to  be  noted  that  cholera  is 
a  disease  of  which  the  onset  and  course  are  much  more  rapid 
than  is  the  case  in  most  infective  diseases,  such  as  typhoid  and 
diphtheria  ;  and  that  recovery  also,  when  it  takes  place,  does  so 
more  quickly.  The  two  factors  to  be  correlated  to  these  facts 
are  (a)  a  rapid  multiplication  of  organisms,  (b)  the  production  of 
rapidly  acting  toxins. 

The  Cholera  Spirillum.  —  Microscopical  Characters.  —  The 
cholera  spirilla  as  found  in  the  intestines  in  cholera  are  small 
organisms  measuring  about  1.5  to  2  /*  in  length,  and  rather  less 
than  .5  in  thickness.  They  are  distinctly  curved  in  one  direc- 
tion, hence  the  appearance  of  a  comma  (Fig.  138);  most  occur 
singly,  but  some  are  attached  in  pairs  and  curved  in  opposite 
directions,  so  that  an  S-shape  results.  Longer  forms  are  rarely 
seen  in  the  intestine,  but  in  cultures  in  fluids,  as  is  especially 
well  seen  in  hanging-drop  preparations,  they  may  grow  into 
longer  spiral  filaments,  showing  a  large  number  of  turns.  If 
film  preparations  be  made  from  the  intestinal  contents  in  typical 


THE   CHOLERA   SPIRILLUM.  409 

cases,  it  will  be  found  that  these  organisms  are  present  in  enor- 
mous numbers  in  almost  pure  culture,  and  that  most  of  the  spi- 
rilla lie  with  their  long  axes  in 
the  same  direction,  so  as  to 
give  the  appearance  which 
Koch  compared  to  a  school  of 
fish  in  a  stream. 

They  possess  very  active 
motility,  which  is  most  marked 
in  the  single  forms.  When 
stained  by  the  suitable  methods 
they  are  seen  to  be  flagellated. 
Usually  a  single  terminal  flagel- 
lum  is  present  at  one  end  only 

(Fig.   I  39).       It  is  Very  delicate,         FlG.  13g.  _  cholera  spirilla,  from  a  culture 

and  measures  four  or  five  times  of  a§ar  of  twenty-four  hours'  growth. 

,_,       ,  i       r    i  Stained  with  weak  carbol-fuchsin.  X  1000. 

the  length  of  the  organism.     In 

some  varieties,  however,  there  may  be  a  flagellum  at  both  ends, 
or  more  than  one  may  be  present ;  cultures  obtained  at  differ- 
ent places  have  shown  considerable  variations  in  this  respect. 


•% 

'  *% 


FlG.    139.  —  Cholera   spirilla   stained  to  FlG.  140.  —  Cholera  spirilla  from  an  old 

show  the  terminal  flagella.     X  1000.  agar  culture,  showing  irregularities  in  size 

and  shape,  with   numerous  faintly  stained 
coccoid  bodies  —  involution  forms. 
Stained  with  fuchsin.     X  1000. 

Cholera  spirilla  do  not  form  spores.  In  old  cultures,  however, 
small,  rounded,  and  highly  refractile  bodies  may  be  found  in  the 
organisms,  which  have  been  regarded  by  Hueppe  as  "arthro- 
spores,"  but  which  are  in  reality  merely  the  result  of  degenera- 


410  CHOLERA. 

tion,  as  they  have  no  higher  powers  of  resistance  than  the 
spirilla  themselves,  and  cultures  containing  enormous  numbers 
of  such  bodies  may  be  found  to  be  quite  dead.  Along  with  such 
appearances  in  old  cultures  there  is  found  great  change  in  the 
size  and  shape  of  the  organisms  (Fig.  140).  Some  are  irregu- 
larly twisted  filaments,  sometimes  globose,  sometimes  clubbed 
at  their  extremities,  and  also  showing  irregular  swellings  along 
their  course.  Others  are  short  and  thick,  and  may  have  the 
appearance  of  large  cocci,  often  staining  faintly.  All  these 
changes  in  appearance  are  to  be  classed  together  as  involution 
forms. 

Staining.  —  Cholera  spirilla  stain  readily  with  the  usual  basic 
aniline  stains,  though  Loffler's  methylene-blue  or  weak  carbol- 
fuchsin  is  specially  suitable.  They  lose  the  stain  in  Gram's 
method. 

Distribution  within  the  Body.  — The  chief  fact  in  this  connec- 
tion is  that  the  spirilla  are  confined  to  the  intestine,  and  are  not 
present  in  the  blood  or  internal  organs.  This  was  determined 
by  Koch  in  his  earlier  work,  and  his  statement  has  been  amply 
confirmed.  In  cases  in  which  there  is  the  characteristic  "  rice- 
water  "  fluid  in  the  intestines,  they  occur  in  enormous  numbers 
—  almost  in  pure  culture.  The  lower  half  of  the  small  intestine 
is  the  part  most  affected.  Its  surface  epithelium  becomes  shed 
in  great  part,  and  the  flakes  floating  in  the  fluid  consist  chiefly 
of  masses  of  epithelial  cells  and  mucus,  amongst  which  are 
numerous  spirilla.  The  spirilla  also  penetrate  the  follicles  of 
Lieberktihn,  and  may  be  seen  lying  between  the  basement 
membrane  and  the  epithelial  lining,  which  becomes  loosened  by 
their  action.  They  are,  however,  rarely  found  in  the  connective 
tissue  beneath,  and  never  penetrate  deeply.  Along  with  these 
changes  there  is  congestion  of  the  mucosa,  especially  around  the 
Peyer's  patches  and  solitary  glands,  which  are  somewhat  swollen 
and  prominent.  In  some  very  acute  cases  the  mucosa  may  show 
general  acute  congestion,  with  a  rosy  pink  colour,  but  very  little 
desquamation  of  epithelium,  the  intestinal  contents  being  a  com- 
paratively clear  fluid  containing  the  spirilla  in  large  numbers. 
In  other  cases  of  a  more  chronic  type,  the  intestine  may  show  more 
extensive  necrosis  of  the  mucosa  and  a  considerable  amount  of 
haemorrhage  into  its  substance,  along  with  formation  of  false 
membrane  at  places.  The  intestinal  contents  in  such  cases  are 


CULTURAL   CHARACTERS.  411 

blood-stained  and  foul-smelling,  there  being  a  great  proportion 
of  other  organisms  present  besides  the  cholera  spirilla  (Koch). 

Cultivation.  —  (For  Methods,  see  p.  426.) 

The  cholera  spirillum  grows  readily  on  all  the  ordinary  media, 
and  with  the  exception  of  that  on  potato,  growth  takes  place  at 
the  ordinary  room  temperature.  The  most  suitable  temperature, 
however,  is  that  of  the  body,  and  growth  usually  stops  about 
16°  C,  though  in  some  cases  it  has  been  obtained  at  a  lower 
temperature. 

Peptone  Gelatin.  —  On  this  medium  the  organism  grows  well 
and  produces  liquefaction.  In  puncture  cultivations  at  22°  C.  a 
whitish  line  appears  along  the  needle  track,  at 
the  upper  part  of  which  liquefaction  commences, 
and  as  evaporation  quickly  occurs,  a  small  bell- 
shaped  depression  forms,  which  gives  the  appear- 
ance of  an  air-bubble.  On  the  fourth  or  fifth  day 
we  get  the  following  appearance  :  There  is  at  the 
surface  the  bubble-shaped  depression  ;  below  this 
there  is  a  funnel-shaped  area  of  liquefaction,  the 
fluid  being  only  slightly  turbid,  but  showing  at 
its  lower  end  thick  masses  of  growth  of  a  more 
or  less  spiral  shape  (Fig.  141).  The  liquefied 
portion  gradually  tapers  off  downwards  towards 
the  needle  track.  (This  appearance  is,  however, 
in  some  varieties  not  produced  till  much  later, 
especially  when  the  gelatin  is  very  stiff,  and,  in 
other  varieties  which  liquefy  very  slowly,  may 
not  be  met  with  at  all.)  At  a  later  stage,  lique- 
faction spreads  and  may  reach  the  side  of  the 
tube. 

In  gelatin  plates  the  colonies  are  somewhat  FIG.  141.— Punc- 
characteristic.  They  appear  as  minute  whitish  ture  culture  of  the 

cholera  spirillum  m 

points,  visible  in  twenty-four  to  forty-eight  hours,  peptone  gelatin  — 
which,  under  a  low  power  of  the  microscope,  do  NaturKzeT0^" 
not  present  a  smooth  circular  outline,  but  one 
which  is  irregularly  granular  or  furrowed ;  as  they  become  larger 
their  surface  has  an  appearance  which  has  been  compared  to 
fragments  of  broken  glass.  Later,  liquefaction  occurs,  and  the 
colony  sinks  into  the  small  cup  formed,  the  plate  then  showing 
small  sharply  marked  rings  around  the  colonies  (Fig.  142). 


412  CHOLERA. 

Under  the  microscope  the  outer  margin  of  the  cup  is  circular 
and  sharply  marked.  Within  the  cup  the  liquefied  portion  forms 
a  ring  which  has  a  more  or  less  granular  appearance,  whilst  the 

mass  of  growth  in  the 
centre  is  irregular  and 
often  broken  up  at  its 
margins.  The  growth 
of  the  colonies  in  gela- 
tin plates  constitutes 
one  of  the  most  impor- 
tant means  of  distin- 

FIG.  142.  —  Colonies  of  the  cholera  spirillum   in  a  rruishing"      the       cholera 
gelatin  plate  ;  three  days'  growth.     A  shows  the  granu-          .    . 

lar  surface,  liquefaction  just  commencing;  in  B,  lique-  Spirillum      from       Other 

faction  is  well  marked.  organisms. 

On  the  surface  of  the  agar  media  a  thin  semi-transparent 
layer  forms,  which  presents  no  special  characters.  On  solidified 
blood  serum  the  growth  has  at  first  the  same  appearance,  but 
afterwards  liquefaction  of  the  medium  occurs.  On  agar  plates 
the  superficial  colonies  under  a  low  power  are  circular  discs  of 
brownish-yellow  colour,  and  more  transparent  than  those  of  most 
other  organisms.  Qi\  potato  at  the  ordinary  temperature,  growth 
does  not  take  place,  but  when  it  is  incubated  at  a  temperature 
of  from  30°  to  37°  C,  a  moist  layer  appears,  which  assumes  a 
dirty  brown  colour  somewhat  like  that  of  the  glanders  bacillus ; 
the  appearance,  however,  varies  somewhat  in  different  varieties, 
and  also  on  different  sorts  of  potatoes. 

In  bouillon  with  alkaline  reaction  the  organism  grows  very 
readily,  there  occurring  in  twelve  hours  at  37°  C.  a  general  tur- 
bidity, while  the  surface  shows  a  thin  pellicle  composed  of  spirilla 
in  a  very  actively  motile  condition.  Growth  takes  place  under 
the  same  conditions  equally  rapidly  in  peptone  solution  (i  per 
cent  with  .5  per  cent  sodium  chloride  added). 

In  milk  also  the  organism  grows  well  and  produces  no  coagu- 
lation nor  any  change  in  its  appearance,  at  least  for  several  days. 

On  all  the  media  the  growth  of  the  cholera  spirillum  is  a 
relatively  rapid  one,  and  especially  is  this  the  case  in  the  peptone 
solution  named  and  in  bouillon,  a  circumstance  of  importance  in 
relation  to  its  separation  in  cases  of  cholera  (vide  p.  426). 

Cholera-red  Reaction.  —  This  is  one  of  the  most  important 
tests  in  the  diagnosis  of  the  cholera  organism.  It  is  always 


THE   CHOLERA-RED   REACTION.  413 

given  by  a  true  cholera  spirillum,  and  though  the  reaction  is  not 
peculiar  to  it,  the  number  of  organisms  which  give  the  reaction 
under  the  conditions  mentioned  are  comparatively  few.  The  test 
is  made  by  adding  one  or  two  drops  of  pure  sulphuric  acid  to  a 
culture  in  peptone  bouillon  or  in  peptone  solution  (i  per  cent) 
which  has  been  incubated  for  twenty-four  hours  at  37°  C.  ;  in 
the  case  of  the  cholera  spirillum  a  reddish-pink  colour  is  pro- 
duced. This  is  due  to  the  fact  that  both  indol  and  a  nitrite  are 
formed  by  the  spirillum  in  the  medium.  The  addition  of  sul- 
phuric acid  causes  a  nitroso-indol  body  to  be  produced  from  these, 
and  this  gives  the  red  colour.  Here,  as  in  testing  for  the  pro- 
duction of  indol  by  other  bacteria,  it  is  found  that  not  every 
specimen  of  peptone  is  suitable,  and  it  is  advisable  to  select  a 
peptone  which  gives  the  characteristic  reaction  with  a  known 
cholera  organism,  and  to  use  it  for  further  tests.  It  is  also 
essential  that  the  sulphuric  acid  should  be  pure,  for  if  traces  of 
nitrites  are  present  the  reaction  might  be  given  by  an  organism 
which  had  not  the  power  of  forming  nitrites. 

The  cholera  organism  is  one  which  grows  much  more  rapidly 
in  the  presence  of  oxygen  than  in  anaerobic  conditions.  Koch, 
in  his  earlier  work,  believed  that  no  growth  took  place  in  the 
absence  of  oxygen,  and  it  has  been  recently  stated  that  this  is 
the  case  in  absolutely  anaerobic  conditions.  Growth,  however, 
takes  place  in  the  ordinary  anaerobic  conditions,  usually  employed 
in  the  culture  of  anaerobic  organisms,  such  as  those  of  tetanus 
and  malignant  oedema,  though  it  occurs  more  slowly  than  in  the 
presence  of  oxygen.  In  the  intestines  the  oxygen  supply,  though 
small  in  amount,  is  yet  sufficient  for  the  growth  of  the  spirilla. 

Powers  of  Resistance.  —  In  their  resistance  against  moist 
heat  cholera  spirilla  correspond  with  most  spore-free  organisms, 
and  are  killed  in  five  minutes  by  a  temperature  of  65°  C.,  and 
much  more  rapidly  at  higher  temperatures.  They  have  com- 
paratively high  powers  of  resistance  against  great  cold,  and  have 
been  found  alive  after  being  exposed  for  several  hours  to  the 
temperature  of  —  10°  C.  They  are,  however,  killed  by  being 
kept  in  ice  for  a  few  days.  Against  the  ordinary  antiseptics 
they  have  comparatively  low  powers  of  resistance,  and  Pfuhl 
found  that  the  addition  of  lime,  in  the  proportion  of  I  per  cent, 
to  water  containing  the  cholera  organisms,  was  sufficient  to  kill 
them  in  the  course  of  an  hour. 


414  CHOLERA. 

As  regards  the  powers  of  resistance  in  ordinary  conditions, 
the  following  facts  may  be  stated.  In  cholera  stools  kept  at  the 
ordinary  room  temperature,  the  cholera  organisms  are  rapidly 
outgrown  by  putrefactive  bacteria,  but  in  some  cases  they  have 
been  found  alive  even  after  two  or  three  months.  In  most 
experiments,  however,  attempts  to  cultivate  them  even  after  a 
much  shorter  time  have  failed.  The  general  conclusion  may  be 
drawn  from  the  work  of  various  observers  that  the  spirilla  do  not 
multiply  freely  in  ordinary  sewage  water,  although  they  may 
remain  alive  for  a  considerable  period  of  time.  In  distilled 
water  they  remain  alive  for  several  weeks  at  least,  but  do  not 
multiply,  nor  does  any  considerable  growth  take  place  without 
the  presence  of  a  pretty  large  proportion  of  organic  matter.  On 
moist  linen,  as  Koch  showed,  they  can  flourish  very  rapidly. 
When  the  cholera  organisms  are  grown  along  with  other  organ- 
isms in  fluids  at  a  warm  temperature,  it  is  found  that  at  first 
they  may  multiply  more  rapidly  than  the  others,  but  that  after 
a  certain  time  they  are  outgrown  by  some  of  the  organisms 
present,  gradually  diminish  in  number,  and  ultimately  disappear. 
It  must  not,  however,  be  inferred  from  such  experiments  that  a 
similar  result  will  necessarily  follow  in  nature,  as  any  particular 
saprophytic  organism  requires  a  special  habitat  —  that  is,  certain 
suitable  conditions  for  its  growth  in  competition  with  other 
organisms.  Though  we  can  state  generally  that  the  conditions 
favourable  for  the  growth  of  the  cholera  spirillum  are,  a  warm 
temperature,  moisture,  a  good  supply  of  oxygen,  and  a  consider- 
able proportion  of  organic  material,  we  do  not  know  the  exact 
circumstances  under  which  it  can  flourish  for  an  indefinite  period 
of  time  as  a  saprophyte.  The  fact  that  the  area  in  which  cholera 
is  an  endemic  disease  is  so  restricted  tends  to  show  that  the 
conditions  for  a  prolonged  growth  of  the  spirillum  outside  the 
body  are  not  usually  supplied.  Yet,  on  the  other  hand,  there 
is  no  doubt  that  in  ordinary  conditions  it  can  live  a  sufficient 
time  outside  the  body  and  multiply  to  a  sufficient  extent,  to 
explain  all  the  facts  known  with  regard  to  the  persistence  and 
spread  of  cholera  epidemics. 

Numerous  experiments  show  that  the  cholera  organisms  are, 
as  a  rule,  rapidly  killed  by  drying,  usually  in  two  or  three  minutes 
when  the  drying  has  been  thorough,  and  it  is  inferred  from  this 
that  they  cannot  be  carried  in  the  living  condition  for  any  great 


INOCULATION   OF   ANIMALS.  415 

distance  through  the  air,  a  conclusion  which  is  well  supported 
by  observations  on  the  spread  of  the  disease.  Cholera  is  practi- 
cally always  transmitted  by  means  of  water  or  food  contaminated 
by  the  organism,  and  there  is  no  doubt  that  contamination  of 
the  water  supply  by  choleraic  discharges  is  the  chief  means  by 
which  areas  of  population  are  rapidly  infected.  It  has  been 
shown  that  if  flies  are  fed  on  material  containing  cholera 
organisms,  the  organisms  may  be  found  alive  within  their 
bodies  twenty-four  hours  afterwards.  And  further,  Haffkine 
found  that  sterilised  milk  might  become  contaminated  with 
cholera  organisms,  if  kept  in  open  jars  to  which  flies  had 
free  access,  in  a  locality  infected  by  cholera.  It  is  quite 
possible  that  infection  may  be  carried  by  this  agency  in  some 
cases. 

Experimental  Inoculation.  —  In  considering  the  effects  of 
inoculation  with  the  cholera  organism,  we  are  met  with  the 
difficulty  that  none  of  the  lower  animals,  so  far  as  is  known,  suf- 
fer from  the  disease  under  natural  conditions.  Even  in  places 
where  cholera  is  endemic,  no  corresponding  affection  has  been 
observed  in  any  animals.  And  further,  before  the  discovery  of 
the  cholera  organism,  various  efforts  had  been  made  to  induce 
the  disease  in  animals  by  feeding  them  with  cholera  dejecta,  but 
without  success.  It  is  therefore  not  surprising  that  the  earlier 
experiments  on  animals  by  feeding  them  with  pure  cultures  were 
attended  with  negative  results.  As  the  organisms  are  confined 
to  the  alimentary  tract  in  the  natural  disease,  attempts  to  induce 
their  multiplication  within  the  intestine  of  animals  by  artificially 
arranging  favouring  conditions,  have  occupied  a  prominent  place 
in  the  experimental  work.  We  shall  give  a  short  account  of 
such  experiments. 

Nikati  and  Rietsch  were  the  first  to  inject  the  organisms  directly  into  the 
duodenum  of  dogs  and  rabbits,  and  they  succeeded  in  producing,  in  a  con- 
siderable proportion  of  the  animals,  a  choleraic  condition  of  the  intestine ;  in 
their  earlier  experiments  the  common  bile  duct  was  ligatured,  but  the  later 
were  performed  without  this  operation.  These  experiments  were  confirmed 
by  other  observers,  including  Koch.  Thinking  that  probably  the  spirillum, 
when  introduced  by  the  mouth,  is  destroyed  by  the  action  of  the  hydrochloric 
acid  of  the  gastric  secretion,  Koch  first  neutralised  this  acidity  by  adminis- 
tering to  guinea-pigs  5  c.c.  of  a  5  per  cent  solution  of  carbonate  of  soda,  and 
some  time  afterwards  introduced  a  pure  culture  into  the  stomach  by  means  of 
a  tube.  Of  nineteen  animals  treated  in  this  way,  only  one  died  with  choleraic 


416  CHOLERA. 

changes  in  the  small  intestine.  This  animal  had  aborted  a  short  time  previ- 
ously, and  as  its  abdominal  walls  were  very  relaxed,  Koch  considered  that 
the  intestinal  peristalsis  had  been  interfered  with,  and  thus  opportunity  had 
been  afforded  to  the  organisms  of  gaining  a  foothold  and  multiplying  in  the 
intestine.  He  accordingly  tried  the  effect  of  artificially  interfering  with  the 
intestinal  peristalsis  by  injecting  tincture  of  opium  into  the  peritoneum  (i  c.c. 
per  200  grm.  weight),  in  addition  to  neutralising  as  before  with  the  carbonate 
of  sodium  solution.  The  result  was  remarkable,  as  thirty  out  of  thirty-five 
animals  treated  died  with  the  same  changes  as  in  the  single  animal  in  the 
previous  series  of  experiments.  The  animals  infected  by  this  method  show 
signs  of  general  prostration,  their  posterior  extremities  being  especially 
weakened  ;  the  abdomen  becomes  tumid,  respiration  slow,  heart's  action  weak, 
and  the  surface  cold.  Death  occurs  after  a  few  hours.  Post  7uorteni  the 
small  intestine  is  distended,  its  mucous  membrane  congested,  and  it  contains 
a  colourless  fluid  with  small  flocculi  and  the  cholera  organisms  in  practically 
pure  cultures.  These  experiments,  which  have  been  repeatedly  confirmed, 
therefore  demonstrated  that  the  cholera  organisms  could,  under  certain  con- 
ditions, set  up  in  animals  a  condition  in  some  respects  resembling  cholera. 
Koch,  however,  found  that  when  the  spirilla  of  Finkler  and  Prior,  of  Deneke, 
and  of  Miller  (vide  infra)  were  employed  by  the  same  method,  a  certain, 
though  much  smaller,  proportion  of  the  animals  died  from  an  intestinal  infec- 
tion. Though  the  changes  in  these  cases  were  not  so  characteristic,  they 
were  sufficient  to  prevent  the  results  obtained  with  the  cholera  organism  from 
being  used  as  a  demonstration  of  the  specific  relation  of  the  latter  to  the 
disease. 

Within  later  years  some  additional  facts  of  high  interest  have  been 
established  with  regard  to  choleraic  infection  of  animals.  For  example, 
Sabolotny  found  that  in  the  marmot  an  intestinal  infection  readily  takes  place 
by  simple  feeding  with  the  organism,  there  resulting  the  usual  intestinal 
changes,  sometimes  with  haemorrhagic  peritonitis,  the  organisms,  however, 
being  present  also  in  the  blood.  It  was  found  by  Issaeff  and  Kolle  that 
young  rabbits  could  be  infected  by  merely  neutralising  the  gastric  secretion 
with  sodium  carbonate,  the  use  of  opium  being  unnecessary ;  but  of  special 
interest  is  the  fact,  discovered  by  Metchnikoff,  that  in  the  case  of  young 
rabbits  shortly  after  birth  a  large  proportion  die  of  choleraic  infection  when 
the  organisms  are  simply  introduced  along  with  the  milk,  as  may  be  done  by 
infecting  the  teats  of  the  mother.  Further,  from  these  animals  thus  infected 
the  disease  may  be  transmitted  to  others  by  a  natural  mode  of  infection.  In 
this  affection  of  young  rabbits  many  of  the  symptoms  of  cholera  are  present 
—  great  prostration,  markedly  subnormal  temperature,  sometimes  anuria,  and 
occasionally  slight  cramps  before  death.  Most  frequently  there  is  diarrhrea, 
though  sometimes  this  may  be  absent,  the  group  of  phenomena  sometimes 
corresponding,  according  to  MetchnjkofF,  with  cholera  sicca.  The  organisms 
occur  in  large  numbers  in  the  intestine,  and  in  some  cases  a  few  may  be  found 
in  the  blood,  and  especially  in  the  gall-bladder.  Many  of  these  experiments 
were  performed  with  the  vibrio  of  Massowah,  which  is  now  admitted  not  to 
be  a  true  cholera  organism,  others  with  a  cholera  vibrio  obtained  from  the 
water  of  the  Seine. 


TOXINS   OF   THE   CHOLERA   SPIRILLUM.  417 

It  will  be  seen  from  the  above  account  that  the  evidence 
obtained  from  experiments  on  intestinal  infection  of  animals, 
though  by  no  means  sufficient  to  establish  the  specific  relation- 
ship of  the  cholera  organism,  is  on  the  whole  favourable  to  this 
view,  especially  when  it  is  borne  in  mind  that  animals  do  not  in 
natural  conditions  surfer  from  the  disease. 

Experiments  performed  by  direct  inoculation  also  supply 
interesting  facts.  Intraperitoneal  injection  in  guinea-pigs  is 
followed  by  general  symptoms  of  illness,  the  most  prominent 
being  distention  of  the  abdomen,  subnormal  temperature,  and, 
ultimately,  profound  collapse.  There  is  peritoneal  effusion, 
which  may  be  comparatively  clear,  or  may  be  somewhat  turbid 
and  contain  flakes  of  lymph,  according  to  the  stage  at  which 
death  takes  place.  If  the  dose  is  large,  organisms  are  found  in 
considerable  numbers  in  the  blood  and  also  in  the  small  intes- 
tine, but  with  smaller  doses  they  are  practically  confined  to  the 
peritoneum.  Kolle  found  that  when  the  minimum  lethal  dose 
was  used  in  guinea-pigs,  the  peritoneum  might  be  free  from 
organisms  at  the  time  of  death,  the  fatal  result  having  taken 
place  from  an  intoxication  (cf.  diphtheria,  p.  365).  These  and 
other  experiments  show  that  though  the  organisms  undergo  a 
certain  amount  of  multiplication  when  introduced  by  the  chan- 
nels mentioned,  still  the  tendency  to  invade  the  tissues  is  not  a 
marked  one.  On  the  other  hand,  the  symptoms  of  general  intox- 
ication are  always  pronounced.  Hence  arise  questions  as  to  the 
nature  and  mode  of  action  of  toxic  bodies  produced  by  the  cholera 
organism. 

Toxins. — Though  there  is  no  doubt  that  there  are  formed 
by  Koch's  spirillum  toxic  bodies  which  produce  many  of  the 
symptoms  of  cholera,  there  is  at  present  very  little  satisfactory 
knowledge  regarding  their  chemical  nature.  The  following 
summary  may  be  given. 

It  has  been  shown,  especially  by  R.  Pfeiffer,1  that  toxic 
phenomena  can  be  produced  by  injection  of  the  dead  spirilla 
into  animals.  A  certain  quantity  of  a  young  culture  on  agar, 
killed  by  exposure  to  the  vapour  of  chloroform,  when  injected 
intraperitoneally  into  a  guinea-pig,  may  cause  death  in  from 

1  Pfeiffer  obtained  his  earlier  results  with  a  vibrio  from  Massowah,  which  is  now 
known  (as  mentioned  above)  not  to  be  a  true  cholera  organism.     The  fact  shows 
that  the  effects  described  are  not  specific  to  the  latter. 
2  E 


418  CHOLERA. 

eight  to  twelve  hours.  There  is  extreme  collapse,  sometimes 
clonic  spasms  occur,  and  the  temperature  may  fall  below  30°  C. 
before  death.  Pfeiffer  considers  that  the  toxic  substances  are 
contained  in  the  bodies  of  the  organisms,  that  is,  they  are 
intracellular,  and  that  they  are  only  set  free  by  the  disintegra- 
tion of  the  latter.  This  opinion  is  grounded  chiefly  on  the  fact 
that  when  bouillon  cultures  were  filtered,  he  found  that  the  fil- 
trate possessed  very  feeble  toxic  properties.  The  dead  cultures 
administered  by  the  mouth  produce  no  effect  unless  the  intes- 
tinal epithelium  is  injured,  in  which  case  poisoning  may  result. 
He  considers  that  the  desquamation  of  the  epithelium  is  an 
essential  factor  in  the  production  of  the  phenomena  of  the 
disease  in  the  human  subject.  Pfeiffer  found  that  the  toxic 
bodies  were  to  a  great  extent  destroyed  at  60°  C.,  but  even  after 
heating  at  100°  C.  a  small  proportion  of  toxin  remained,  which 
had  the  same  physiological  action. 

On  the  other  hand,  other  observers  (Petri,  Ransom,  Klein, 
and  others)  have  obtained  toxic  bodies  from  filtered  cultures. 
Metchnikoff,  E.  Roux,  and  Taurelli-Salimbeni  have  demon- 
strated the  formation  of  such  diffusible  toxic  bodies  in  fluid 
media  in  the  following  manner.  Small  collodion  sacs  were  pre- 
pared, each  containing  2  to  4  c.c.  of  bouillon.  One  sac  was 
inoculated  with  a  living  virulent  culture  of  the  cholera  vibrio  ; 
to  the  second,  two  entire  cultures  on  agar  of  the  same  organism 
were  added,  the  cultures  being  first  killed  by  chloroform.  Each 
sac  was  then  closed  and  placed  with  aseptic  precautions  in  the 
peritoneum  of  a  guinea-pig.  The  animal  which  received  the  sac 
containing  the  living  vibrios  soon  showed  symptoms  of  choleraic 
poisoning,  and  died  in  a  few  days,  whilst  the  animal  which 
received  the  sac  containing  large  quantities  of  dead  organisms 
showed  only  transitory  symptoms  of  illness.  These  observers 
therefore  concluded  that  toxic  substances  are  formed  by  the 
living  organisms,  which  quickly  diffuse  into  the  medium  (and  in 
the  experiments,  through  the  wall  of  the  sac).  By  greatly 
increasing  the  virulence  of  the  organism,  then  growing  it  in 
bouillon  and  filtering  the  cultures  on  the  third  and  fourth  day, 
they  obtained  a  fluid  which  was  highly  toxic  to  guinea-pigs  (the 
fatal  dose  usually  being  \  c.c.  per  100  grm.  weight).  If  the 
dose  of  the  toxin  is  very  large,  death  follows  in  an  hour  or  even 
less.  The  symptoms  closely  resemble  those  obtained  by  Pfeiffer, 


HUMAN    EXPERIMENTS. 


419 


the  rapid  fall  of  temperature  being  a  striking  feature.  They 
found  that  the  toxicity  of  the  filtrate  was  not  altered  by  boiling. 
It  is  somewhat  difficult  to  reconcile  the  results  of  Pfeiffer  and 
Metchnikoff  as  regards  the  action  of  heat,  and  there  is  evidence 
that  the  toxic  substances  present  in  the  bodies  of  the  spirilla 
differ  from  those  present  in  filtered  cultures.  A  considerable 
number  of  observers,  however,  agree  in  stating  that,  at  least, 
some  toxins  obtained  from  cholera  cultures  are  not  destroyed  at 
100°  C. 

A  great  many  observers  have  attempted  to  obtain  toxins  in  a  chemically 
pure  condition,  but  so  far  without  results  which  can  be  regarded  as  conclusive. 
Hueppe  and  Wood  found  that  the  most  active  toxins  were  produced  by 
growing  the  cholera  organism  in  albumin  in  anaerobic  conditions,  and  con- 
sidered that  this  corresponded  to  the  mode  of  their  production  in  cholera. 
Scholl  confirmed  Hueppe's  results,  and  obtained  from  cultures  under  such 
conditions  a  peptone  which  possessed  highly  toxic  properties,  and  which  he 
called  cholera  toxo-peptone.  These  results,  however,  have  been  adversely 
criticised  by  various  observers.  Wesbrook  obtained  different  substances 
according  to  the  media  on  which  the  cholera  organisms  were  grown,  and  yet 
these  produced  very  much  the  same  effects,  chiefly  collapse,  subnormal 
temperature,  cramps,  dyspnrea>  etc.  Such  toxic  bodies  were  even  obtained 
from  cultures  in  asparaginate  of  soda  solution,  which  did  not  contain  any 
proteid  substance.  He  therefore  came  to  the  conclusion  that  the  so-called 
toxalbumins,  etc.,  are  really  mixtures  of  albumins  and  true  toxins,  the 
chemical  nature  of  the  latter  not  having  been  yet  determined.  Wesbrook 
also  obtained  the  toxic  bodies  in  small  quantity  from  the  pleural  exudate  of 
guinea-pigs  killed  by  the  vibrio.  Bosc  also  found  that  the  blood,  and  to  a. 
less  extent  the  urine,  of  patients  who  had  died  in  the  algid  stage,  produce 
toxic  phenomena  and  death,  when  injected  intravenously  in  rabbits.  In  this 
case  also,  nothing  is  known  with  regard  to  the  chemical  nature  of  the  toxic 
bodies. 

Experiments  on  the  Human  Subject.  —  Experiments  have  also 
been  performed  in  the  case  of  the  human  subject,  both  intention- 
ally and  accidentally.  In  the  course  of  Koch's  earlier  work,  one 
of  the  workers  in  his  laboratory  shortly  after  leaving  was  seized 
with  severe  choleraic  symptoms.  The  stools  were  found  to 
contain  cholera  spirilla  in  enormous  numbers.  Recovery,  how- 
ever, took  place.  In  this  case  there  was  no  other  possible 
source  of  infection  than  the  cultures  with  which  the  man  had 
been  working,  as  no  cholera  was  present  in  Germany  at  the  time. 
Within  recent  years  a  considerable  number  of  experiments  have 
been  performed  on  the  human  subject,  which  certainly  show  that 
in  some  cases  more  or  less  severe  choleraic  symptoms  may  follow 


420  CHOLERA. 

ingestion  of  pure  cultures,  whilst  in  others  no  effects  may  result. 
The  former  was  the  case,  for  example,  with  Emmerich  and 
Pettenkofer,  who  made  experiments  on  themselves,  the  former 
especially  becoming  seriously  ill.  In  the  case  of  both,  diarrhoea 
was  well  marked,  and  numerous  cholera  spirilla  were  present  in 
the  stools,  though  toxic  symptoms  were  proportionately  little 
pronounced.  Metchnikoff  also,  by  experiments  on  himself  and 
others,  obtained  results  which  convinced  him  of  the  specific  re- 
lation of  the  cholera  spirillum  to  the  disease.  Lastly,  we  may 
mention  the  case  of  Dr.  Orgel  in  Hamburg,  who  contracted  the 
disease  in  the  course  of  experiments  with  the  cholera  and  other 
spirilla,  and  died  in  spite  of  treatment.  It  is  believed  that  in 
sucking  up  some  peritoneal  fluid  containing  cholera  spirilla,  a 
little  entered  his  mouth  and  thus  infection  was  produced.  This 
took  place  in  September  1894  at  a  time  when  there  was  no 
cholera  in  Germany.  On  the  other  hand,  in  many  cases  the 
experimental  ingestion  of  cholera  spirilla  by  the  human  subject 
has  given  negative  results.  Still,  as  the  result  of  observation  of 
what  takes  place  in  a  cholera  epidemic,  it  is  the  general  opinion 
of  authorities  that  only  a  certain  proportion  of  people  are 
susceptible  to  cholera,  and  the  facts  mentioned  above  have,  in 
our  opinion,  great  weight  in  establishing  the  relation  of  the 
organism  to  the  disease. 

Immunity.  — As  this  subject  is  discussed  later,  only  a  few 
facts  will  be  here  stated,  chiefly  for  the  purpose  of  making  clear 
what  follows  with  regard  to  the  means  of  distinguishing  the 
cholera  spirillum  from  other  organisms.  The  guinea-pig  or  any 
other  animal  may  be  easily  immunised  against  the  cholera 
organism  by  repeated  injections  (conveniently  made  into  the 
peritoneum)  of  non-fatal  doses  of  the  spirilla.  It  is  better  to 
commence  the  process  with  non-fatal  doses  of  cultures  killed  by 
the  vapour  of  chloroform  or  by  heat,  the  doses  being  gradually 
increased,  and  afterwards  to  proceed  with  increasing  doses  of 
the  living  organism.  In  this  way  a  high  degree  of  immunity 
against  the  organism  is  developed,  and  further,  the  blood 
serum  of  an  animal  thus  immunised  (anti-cholera  serum) 
has  markedly  protective  power  when  injected,  even  in  a  small 
quantity,  into  a  guinea-pig  along  with  five  or  ten  times  the 
fatal  dose  of  the  living  organism  ;  it  has  also  the  property 
of  agglutinating  the  cholera  organism.  These  properties, 


IMMUNITY   AGAINST   CHOLERA. 


421 


being  within  certain  limits  specific,  constitute  an  additional  aid 
in  the  diagnosis  of  an  organism  supposed  to  be  the  cholera 
spirillum. 

A  curious  fact,  however,  is,  that  immunity  produced  by  the  above  method 
is  only  exerted  against  the  living  organisms,  and  does  not  protect  against  the 
toxins  —  that  is,  it  is  due  to  certain  substances  which  act  as  germicides  (indi- 
rectly), but  which  are  not  antitoxic.  Further,  it  does  not  protect  the  guinea- 
pig  from  the  intestinal  infection  by  Koch's  method  (Pfeiffer  and  Wassermann, 
Sobernheim.  Klein),  nor  does  the  anti-cholera  serum  protect  young  rabbits 
against  the  choleraic  affection  produced  by  ingestion  of  the  cholera  vibrios 
(Metchnikoff).  The  inference  from  these  latter  results  appears  to  be,  that 
when  the  vibrios  are  introduced  into  the  tissues,  they  are  killed  by  certain 
substances  in  the  serum,  but  in  the  intestine  they  are  in  a  sense  outside  of  the 
tissues,  and  can  there  multiply  and  produce  toxins.  Metchnikoff  has  prepared 
a  true  antitoxic  cholera  serum  by  injections  of  repeated  and  gradually  increased 
doses  of  the  toxin,  and  has  found  that  this  antitoxic  serum  has  a  distinct  effect 
against  the  choleraic  affection  of  rabbits. 

Anti-cholera  Inoculation. — Haffkine's  method  for  inoculation 
against  cholera  exemplifies  the  above  principles.  It  depends 
upon  (a)  attenuation  of  the  virus  —  that  is,  the  cholera  organism, 
and  (b]  exaltation  of  the  virus.  The  virulence  of  the  organism 
is  diminished  by  passing  a  current  of  sterile  air  over  the  surface 
of  the  cultures,  or  by  various  other  methods.  The  virulence  is 
exalted  by  the  method  Qi passage  —  that  is,  by  growing  the  organ- 
ism in  the  peritoneum  in  a  series  of  guinea-pigs.  By  the  latter 
method  the  virulence  after  a  time  is  increased  twenty -fold  — that 
is,  the  fatal  dose  has  been  reduced  to  a  twentieth  of  the  original. 
Cultures  treated  in  this  way  constitute  the  virus  exalte.  Subcu- 
taneous injection  of  the  virus  exalte  produces  a  local  necrosis, 
and  may  be  followed  by  the  death  of  the  animal,  but  if  the 
animal  be  treated  first  with  the  attenuated  virus,  the  subsequent 
injection  of  the  virus  exalte  produces  only  a  local  oedema.  After 
inoculation  first  by  attenuated  and  afterwards  by  exalted  virus, 
the  guinea-pig  has  acquired  a  high  degree  of  immunity,  and 
Haffkine  believed  that  this  immunity  was  effective  in  the  case 
of  every  method  of  inoculation,  that  is,  by  the  mouth  as  well  as 
by  injection  into  the  tissues.  After  trying  his  method  on  the 
human  subject  and  finding  it  free  from  risk,  he  extended  it  in 
practice  on  a  large  scale  in  India  in  1894,  and  these  experiments 
are  still  going  on.  So  far  the  results  are,  on  the  whole,  distinctly 
encouraging.  In  the  human  subject  two  or  sometimes  three  in- 


422  CHOLERA. 

oculations  are  made  with  attenuated  virus  before  the  virus  exalte 
is  used.  Wassermann  and  Pfeiffer,  and  also  Klein,  have  found, 
however,  that  guinea-pigs  immunised  by  Haffkine's  method  are 
not  immunised  against  intestinal  infection  when  the  animal  is 
treated  by  Koch's  method  (that  is,  by  paralysing  the  intestines 
with  opium,  vide  p.  416).  Notwithstanding  this  fact  Haffkine's 
method  may  still  have  a  beneficial  effect,  though  it  may  not  be 
preventive  in  all  cases. 

Means  of  distinguishing  the  Cholera  Organism.  —  According 
to  Koch  the  most  important  points  in  the  diagnosis  of  cholera 
are :  — 

(a)  Microscopical  characters  of  the  dejecta,  (b)  Appearance 
of  the  colonies  in  gelatin  plates,  (c)  Their  appearance  on  agar 
plates,  (d)  The  growth  in  peptone  solution,  (e)  The  cholera- 
red  reaction.  (/)  The  effect  of  intraperitoneal  inoculation  of 
guinea-pigs  with  pure  cultures. 

There  can  be  no  doubt  that  in  the  great  majority  of  cases 
these  points  taken  collectively  are  sufficient  in  identifying  the 
cholera  organism.  In  addition,  however,  the  various  properties 
of  an  anti-cholera  serum  may  be  employed  ;  of  these  the  most 
easily  applied  is  the  agglutinative  reaction.  The  following  is  an 
account  of  Pfeiffer's  reaction,  which  was  the  first  to  be  intro- 
duced. 

Pfeiffer^s  Reaction.  — A  loopful  (2  mgrm.)  of  recent  agar  culture  of  the 
organism  to  be  tested  is  added  to  i  c.c.  of  ordinary  bouillon  containing  .001 
c.c.  of  anti-cholera  serum.  The  mixture  is  then  injected  into  the  peritoneal 
cavity  of  a  young  guinea-pig  (about  200  grm.  in  weight),  and  the  peritoneal 
fluid  of  this  janimal  (conveniently  obtained  by  means  of  capillary  glass  tubes 
inserted  into  the  peritoneum)  is  examined  microscopically  after  a  few  minutes. 
If  the  spirilla  injected  have  been  cholera  spirilla,  it  will  be  found  that  they 
become  motionless,  swell  up  into  globules,  and  ultimately  break  down  and 
disappear — positive  residt.  If  they  are  found  active  and  motile,  then  the  pos- 
sibility of  their  being  true  cholera  spirilla  may  be  excluded  —  negative  result. 
In  the  former  case  (positive  result)  there  is,  however,  still  the  possibility  that 
the  organism  is  devoid  of  pathogenic  properties  and  has  been  destroyed  by 
the  normal  peritoneal  fluid.  A  control  experiment  should  accordingly  be 
made  with  .001  c.c.  of  normal  serum  in  place  of  the  anti-cholera  serum.  If 
no  alteration  of  the  organism  occurs  with  its  use,  then  it  is  to  be  concluded 
that  the  organism  in  question  has  been  demonstrated  by  the  specific  reaction 
to  be  the  cholera  spirillum  (see  Chapter  XX.). 

Properties  of  the  Serum  of  Cholera  Patients  and  Conva- 
lescents. —  Lazarus  was  the  first  to  show  that  the  serum  of 


PROPERTIES   OF   IMMUNE    SERUM. 


423 


patients  who  had  suffered  from  cholera  possessed  the  power  of 
protecting  guinea-pigs,  when  injected  in  very  minute  quantity 
along  with  a  fatal  dose  of  the  cholera  organism.  These  experi- 
ments were  confirmed  by  Klemperer,  Issaeff,  and  Pfeiffer,  and 
the  last  mentioned  found  that  the  serum  of  such  patients  gave 
the  characteristic  reaction  if  injected  with  the  spirilla  into  the 
peritoneal  cavity  of  a  guinea-pig.  Further,  so  far  as  experiment 
has  gone,  this  action  is  not  exerted  against  any  other  organism 
—  that  is,  it  is  specific  towards  the  cholera  spirillum.  This 
action  of  the  serum  may  be  present  eight  or  ten  days  after  the 
attack  of  the  disease,  but  is  most  marked  four  weeks  after ;  it 
then  gradually  becomes  weaker  and  disappears  in  two  or  three 
months  (Pfeiffer  and  Issaeff). 

Specific  agglutinative  properties  have,  however,  been  detected 
in  the  serum  of  cholera  patients  at  a  much  earlier  date,  in  some 
cases  even  on  the  first  day  of  the  disease,  though  usually  a  day 
or  two  later.  The  dilutions  used  were  1-15  to  1-120,  and  these 
had  no  appreciable  effect  on  organisms  other  than  the  cholera 
spirillum  (Achard  and  Bensande).  Needless  to  say,  such  facts 
supply  strong  additional  evidence  of  the  relation  of  Koch's 
spirillum  to  cholera. 

General  Summary. — We  may  briefly  summarise  as  follows 
the  facts  in  favour  of  Koch's  spirillum  being  the  cause  of  cholera. 
First,  there  is  the  constant  presence  of  spirilla  in  true  cases 
of  cholera,  which  on  the  whole  conform  closely  with  Koch's 
description,  though  variations  undoubtedly  occur.  Moreover, 
the  facts  known  with  regard  to  their  conditions  of  growth,  etc., 
are  in  conformity  with  the  origin  and  spread  of  cholera  epidemics. 
Secondly,  the  experiments  on  animals  with  Koch's  spirillum  or 
its  toxins  give  as  definite  results  as  one  can  reasonably  look  for 
in  view  of  the  fact  that  animals  do  not  suffer  naturally  from  the 
disease.  Jliirdly,  the  experiments  on  the  human  subject  and  the 
results  of  accidental  infection  by  means  of  pure  cultures  are  also 
strongly  in  favour  of  this  view.  Fourthly,  the  agglutinative  and 
protective  properties  of  the  serum  of  cholera  patients  and  con- 
valescents constitute  another  point  in  its  favour.  Fifthly,  bac- 
teriological methods,  which  proceed  on  the  ' assumption  that 
Koch's  spirillum  is  the  cause  of  the  disease,  have  been  of  the 
greatest  value  in  the  diagnosis  of  the  disease.  And  lastly,  the 
results  of  Haffkine's  method  of  preventive  inoculation  in  the 


424  CHOLERA. 

human  subject,  which  are  on  the  whole  favourable,  also  supply 
additional  evidence.  If  all  these  facts  are  taken  together,  we 
consider  the  conclusion  must  be  arrived  at  that  the  growth  of 
Koch's  spirillum  in  the  intestine  is  the  immediate  cause  of  the 
disease.  This  does  not  exclude  the  probability  of  an  important 
part  being  played  by  conditions  of  weather  and  locality,  though 
such  are  very  imperfectly  understood.  Pettenkofer,  for  example, 
recognised  two  main  factors  in  the  causation  of  epidemics,  which 
he  designated  x  and  y,  and  considered  that  these  two  must  be 
present  together  in  order  that  cholera  may  spread.  The  x  is  the 
direct  cause  of  the  disease  —  an  organism  which  he  admitted 
to  be  Koch's  spirillum  ;  the  y  includes  climatic  and  local  con- 
ditions, e.g.,  state  of  ground-water,  etc. 

Difficulty  does  not  arise,  however,  so  much  with  regard  to 
the  causal  relationship  of  Koch's  spirillum  to  cholera  as  in 
connection  with  various  organisms  which  have  been  culti- 
vated from  other  sources,  and  which  more  or  less  closely 
resemble  it. 

Other  Spirilla  resembling  the  Cholera  Organism.  —  These 
have  been  chiefly  obtained  either  from  water  contaminated  by 
sewage  or  from  the  intestinal  discharge  in  cases  with  choleraic 
symptoms.  Some  of  them  differ  so  widely  in  their  cultural  and 
other  characters  (some,  for  example,  are  phosphorescent)  that  no 
one  would  hesitate  to  classify  them  as  distinct  species.  Others, 
however,  closely  resemble  the  cholera  organism. 

The  vibrio  berolinensis,  cultivated  by  Neisser  from  Berlin  sewage  water, 
differs  from  the  cholera  organism  only  in  the  appearance  of  its  colonies  in 
gelatin  plates,  its  weak  pathogenic  action,  and  its  giving  a  negative  result  with 
Pfeiffer's  test.  It,  however,  gives  the  cholera-red  reaction.  The  vibrio  Danubi- 
cus,  cultivated  by  H eider  from  canal  water,  also  differs  in  the  appearance  of  its 
colonies  in  plates,  and  also  reacts  negatively  to  Pfeiffer's  test ;  in  most  respects 
it  closely  resembles  the  cholera  organism.  Another  spirillum  (v.  Ivanoff) 
was  cultivated  by  Ivanoff  from  the  stools  of  a  typhoid  patient  after  these  had 
been  diluted  with  water.  This  organism  differed  somewhat  in  the  appearance 
of  its  colonies  and  in  its  great  tendency  to  grow  out  in  the  form  of  long 
threads,  but  Pfeiffer  found  that  it  reacted  to  his  test  in  the  same  way  as  the 
cholera  organism,  and  he  considered  that  it  was  really  a  variety  of  the  cholera 
organism.  No  spirilla  could  be  found  microscopically  in  the  stools  in  this 
case,  and  Pfeiffer  is  of  the  opinion  that  the  organism  gained  entrance  acci- 
dentally. These  examples  will  show  how  differences  of  opinion,  even  amongst 
experts,  might  arise  as  to  whether  a  certain  spirillum  were  really  the  cholera 
organism  or  a  distinct  species  resembling  it. 


SPIRILLA   RESEMBLING   CHOLERA   SPIRILLUM.          425 

A  few  'examples  may  also  be  given  of  organisms  cultivated 
from  cases  in  which  cholera-like  symptoms  were  present. 

The  vibrio  of  Massowah  was  cultivated  by  Pasquale  from  a  case  during 
a  small  epidemic  of  cholera.  This  organism  so  closely  resembles  Koch's 
spirillum  that  it  was  accepted  by  several  authorities  as  the  true  cholera  organ- 
ism, and,  as  already  stated,  Metchnikoff  produced  by  it  cholera  symptoms  in 
the  human  subject,  and  also  the  cholera-like  disease  in  young  rabbits.  It  pos- 
sesses four  flagella,  has  a  high  degree  of  virulence,  producing  septicaemia  both 
in  guinea-pigs  and  pigeons,  and  its  colonies  in  plates  differ  somewhat  from  the 
cholera  organism.  Moreover,  it  reacts  negatively  to  Pfeiffer's  test.  Another 
organism,  the  v,  Gindha,  was  cultivated  by  Pasquale  from  a  well,  and  was  at 
first  accepted  by  Pfeiffer  as  the  cholera  organism,  but  afterwards  rejected, 
chiefly  because  it  failed  to  give  the  specific  immunity  reaction.  It  also  differs 
somewhat  from  the  cholera  organism  in  its  pathogenic  effects,  and  it  fails  to 
give  the  cholera-red  reaction  or  gives  it  very  faintly. 

Pestana  and  Bettencourt  also  cultivated  a  species  of  spirillum  from  a  num- 
ber of  cases  during  an  epidemic  in  Lisbon  —  an  epidemic  in  which  there  were 
symptoms  of  gastro-enteritis,  although  only  in  a  few  instances  did  the  disease 
resemble  cholera.  They  also  cultivated  the  same  organism  from  the  drinking 
water.  It  differs  from  the  cholera  organism  in  the  appearance  of  its  colonies 
and  of  puncture  cultures  in  gelatin.  It  has  very  feeble  pathogenic  effects,  and 
gives  a  very  faint,  or  no,  cholera-red  reaction.  To  Pfeiffer's  test  it  also  reacts 
negatively.  Another  spirillum  (v.  Romamis}  was  obtained  by  Celli  and  San- 
tori  from  twelve  out  of  forty-four  cases  where  there  were  the  symptoms  of  mild 
cholera.  This  organism  does  not  give  the  cholera-red  reaction,  nor  is  it  patho- 
genic for  animals.  They  look  upon  it  as  a  "  transitory  variety  "  of  the  cholera 
organism,  though  sufficient  evidence  for  this  view  is  not  adduced. 

We  have  mentioned  these  examples  in  order  to  show  some  of 
the  difficulties  which  exist  in  connection  with  this  subject.  It  is 
important  to  note  that,  on  the  one  hand,  spirilla  which  have 
been  judged  to  be  of  different  species  from  the  cholera  organ- 
ism have  been  cultivated  from  cases  in  which  cholera-like 
symptoms  were  present,  and,  on  the  other  hand,  in  cases  of 
apparently  true  cholera  considerable  variations  in  the  characters 
of  the  cholera  organisms  have  been  found.  Such  variations 
have  especially  been  recorded  by  Surgeon-major  Cunningham 
in  India.  It  is  therefore  quite  an  open  question  whether  some 
of  the  organisms  in  the  former  case  may  not  be  cholera  spirilla 
which  have  undergone  variations  as  a  result  of  the  conditions  of 
their  growth.  That  such  variations  may  occur  we  have  a  con- 
siderable- amount  of  evidence.  The  great  bulk  of  evidence, 
however,  goes  to  show  that  Asiatic  cholera  always  spreads  as 
an  epidemic  from  places  in  India  where  the  disease  is  endemic, 


426  CHOLERA. 

and  that  its  direct  cause  is  Koch's  spirillum.  It  is  sufficient  to 
bear  in  mind  that  choleraic  symptoms  may  be  produced  by  other 
causes,  and  that  in  some  of  such  cases  spirilla  which  have  some 
resemblance  to  Koch's  organism  mayi  be  present  in  the  intes- 
tinal discharges,  though  rarely  in  large  numbers. 

Methods  of  Diagnosis.  —  In  the  first  place,  the  stools  ought 
to  be  examined  microscopically.  Dried  film  preparations  should 
be  made  and  stained  by  any  ordinary  stains,  though  carbol-fuch- 
sin  diluted  four  times  with  water  is  specially  to  be  recommended. 
Hanging-drop  preparations,  with  or  without  the  addition  of  a 
weak,  watery  solution  of  gentian-violet  or  other  stain,  should  also 
be  made,  by  which  method  the  motility  of  the  organism  can  be 
readily  seen.  By  microscopic  examination  the  presence  of  spirilla 
will  be  ascertained,  and  an  idea  as  to  their  number  obtained. 
In  some  cases  the  cholera  spirilla  are  so  numerous  in  the  stools 
that  a  picture  is  presented  which  is  obtained  in  no  other  condi- 
tion, and  a  microscopic  examination  may  be  sufficient  for  prac- 
tical purposes.  According  to  Koch,  a  diagnosis  was  made  in 
50  per  cent  of  the  cases  during  the  Hamburg  epidemic  by  micro- 
scopic examination  alone.  In  the  case  of  the  first  appearance 
of  a  cholera-like  disease,  however,  all  the  other  tests  should  be 
applied  before  a  definite  diagnosis  of  cholera  is  made. 

If  the  organisms  are  very  numerous,  gelatin  or  agar  plates 
may  be  made  at  once  and  pure  cultures  obtained. 

Schottelius*  Enriching  Method.  —  If  the  spirilla  occur  in  com- 
paratively small  numbers,  the  best  method  is  to  inoculate  pep- 
tone solution  ( i  per  cent)  and  incubate  for  eight  to  twelve  hours. 
At  the  end  of  that  time  the  spirilla  will  be  found  on  microscopic 
examination  in  enormous  numbers  at  the  surface,  and  thereafter 
plate  cultures  can  readily  be  made.  If  the  spirilla  are  very  few 
in  number,  or  if  a  suspected  water  is  to  be  examined  for  cholera 
organisms,  the  peptone  solution  which  has  been  inoculated  should 
be  examined  at  short  intervals  till  the  spirilla  are  found  micro- 
scopically. A  second  flask  of  peptone  solution  should  then  be 
inoculated,  and  possibly  again  a  third  from  the  second.  By  this 
method,  properly  carried  out,  a  culture  may  be  obtained  which, 
though  impure,  contains  a  large  proportion  of  the  spirilla,  and 
then  plate  cultures  may  be  made. 

When  a  spirillum  has  been  obtained  in  pure  condition  by 
these  methods,  the  appearance  of  the  colonies,  in  plates  should 


SPIRILLUM    METCHNIKOVI.  427 

be  specially  noted,  the  test  for  the  cholera-red  reaction  should 
be  applied,  and  in  many  cases  it  is  advisable  to  test  the  effects 
of  intraperitoneal  injection  of  a  portion  of  a  recent  agar  culture 
in  a  guinea-pig,  the  amount  sufficient  to  cause  death  being  also 
ascertained.  The  agglutinating  or  sedimenting  properties  of  the 
serum  of  the  patient  should  be  tested  against  a  known  cholera 
•organism,  and  against  the  spirillum  cultivated  from  the  case.  In 
the  same  way  the  action  of  the  serum  of  an  immunised  guinea- 
pig  may  be  tested  both  as  regards  agglutinating  and  protective 
properties. 

A  number  of  other  spirilla  have  been  cultivated,  which  are  of 
interest  on  account  of  their  points  of  resemblance  to  the  cholera 
organism,  though  probably  they  produce  no  pathological  condi- 
tions in  the  human  subject. 

Metchnikoff's  Spirillum  (vibrio  Metchnikovi) .  —  This  organism  was  ob- 
tained by  Gamaleia  from  an  epidemic  disease  of  fowls  in  Odessa,  and  is  of 
special  interest  on  account  of  its  close 

resemblance  to  the  cholera  organism.  •  ^f,y£\     V 

In  the  natural  disease,  which  especially  '  >  £>  *     A  «.* 

affects  young  fowls, the  animals  suffer  *  i  ^  JS 

from  diarrhrea,  pass  into  a  sort  of 
stupor,  sitting  with  their  feathers 
ruffled,  and  usually  die  within  forty- 
eight  hours.  The  intestines  contain 
a  greyish-yellow  fluid,  sometimes 
slightly  blood-stained,  in  which  the 
spirilla  are  found.  A  few  of  these 
spirilla  may  also  be  found  in  the 
blood  in  the  younger  fowls,  though 
generally  absent  from  the  blood  in 
the  older. 

Morphologically  the  organism  is        FIG.  143.— Metchnikoff's  spirillum,  both 

practically  identical  with  Koch's  spiril-   in  ,curvedf  and  strfaigh<  forms ;  fro™  an  agar 
3  F  culture  of  twenty-four  hours  growth. 

lum  (Fig.  143).  It  is  actively  motile,  stained  with  weak  carbol-fuchsin.  x  TOCO. 
and  has  the  same  staining  reactions. 

Its  growth  in  peptone  gelatin  also  closely  resembles  that  of  the  cholera  organ- 
ism, though  it  produces  liquefaction  more  rapidly  (Fig.  144,  A).  In  gelatin 
plates  the  young  colonies  are,  however,  smoother  and  more  circular.  After 
liquefaction  occurs,  some  of  the  colonies  are  almost  identical  in  appearance  with 
those  of  the  cholera  organism,  whilst  others  show  more  uniformly  turbid  con- 
tents. In  puncture  cultures  the  growth  takes  place  more  rapidly,  but  in  ap- 
pearance closely  resembles  that  of  the  cholera  organism  a  few  days  older.  Its 
growth  in  peptone  solution,  too,  is  closely  similar,  and  it  also  gives  the  cholera- 
red  reaction. 


<$*£SfflF*&. 

.     -KW,**  .  ^v.          9f  , 


428 


CHOLERA. 


This  organism  can,  however,  be  readily  distinguished  from  the  cholera, 
organism  by  the  effects  of  inoculation  on  animals,  especially  on  pigeons  and 

guinea-pigs.  Subcutaneous  inoculation  of  small 
quantities  of  pure  culture  in  pigeons  is  followed 
by  septicaemia  which  produces  a  fatal  result 
usually  within  twenty-four  hours.  Inoculation 
with  the  same  quantity  of  cholera  organism  pro- 
duces practically  no  result ;  even  with  large 
quantities  death  is  rarely  produced.  The  vibrio 
Metchnikovi  produces  somewhat  similar  effects 
in  the  guinea-pig  to  those  in  the  pigeon,  sub- 
cutaneous inoculation  being  followed  by  exten- 
sive haemorrhagic  oedema,  and  a  rapidly  fatal 
septicaemia.  Young  fowls  can  be  infected  by 
feeding  with  virulent  cultures.  We  have  evi- 
dence from  the  work  of  Gamaleia  that  the  toxins 
of  this  organism  have  somewhat  the  same  action 
as  those  of  the  cholera  organism. 

The  organism  is  therefore  one  which  very 
closely  resembles  the  cholera  organism,  the 
results  on  inoculating  the  pigeon  offering  the 
most  ready  means  of  distinction.  It  gives  a 
negative  reaction  to  Pfeiffer's  test ;  that  is,  the 
properties  of  an  anti-cholera  serum  are  not 
exerted  against  it.  It  may  also  be  mentioned 
that  an  organism  which  is  apparently  the  same 
as  the  vibrio  Metchnikovi  was  cultivated  by 
Pfuhl  from  water,  and  named  V.  Nordhafen. 

Spirillum  Schuylkiliensis.  —  A  spirillum 
similar  in  its  cultural  characters  and  pathogenic 
properties  to  Sp.  Metchnikovi  has  been  isolated 
from  the  waters  of  the  Schuylkill  river  at  Philadelphia,  by  Abbott,  and  termed 
by  him  spirillum  Schuylkiliensis. 

Finkler  and  Prior's  Spirillum. — These  observers,  shortly  after  Koch's 
discovery  of  the  cholera  organism,  separated  a  spirillum,  in  a  case  of  cholera 
nostras,  from  the  stools  after  they  had  been  allowed  to  decompose  for  several 
days.  There  is,  however,  no  evidence  that  the  spirillum  has  any  causal 
relationship  to  this  or  any  other  disease  in  the  human  subject.  Morphologi- 
cally it  closely  resembles  Koch's  spirillum,  and  cannot  be  distinguished  from 
it  by  its  microscopical  characters,  although,  on  the  whole,  it  tends  to  be  rather 
thicker  in  the  centre  and  more  pointed  at  the  ends  (Fig.  145).  In  cultures, 
however,  it  presents  marked  differences.  In  puncture  cultures  on  gelatin  it 
grows  much  more  quickly,  and  liquefaction  is  generally  visible  within  twenty- 
four  hours.  The  liquefaction  spreads  rapidly,  and  usually  in  forty-eight  hours 
it  has  produced  a  funnel-shaped  tube  with  turbid  contents,  denser  below 
(Fig.  144,  B).  In  plate-cultures  the  growth  of  the  colonies  is  proportionately 
rapid.  Before  they  have  produced  liquefaction  around  them,  they  appear,  un- 
like those  of  the  cholera  organism,  as  minute  spheres  with  smooth  margins.. 


A  B 

FIG.  144.  —  Puncture  cultures 
in  peptone  gelatin. 

A.  Metchnikoff 's  spirillum.    Five 
days'  growth. 

B.  Finkler  and  Prior's   spirillum. 
Four  days'  growth.     Natural  size. 


FINKLER   AND   PRIOR'S    SPIRILLUM. 


429 


When  liquefaction  occurs,  they  appear  as  little  spheres  with  turbid  contents, 

which  rapidly  increase  in  size  ;   ultimately  general  liquefaction  occurs.     On 

potatoes  this  organism  grows  well  at  the  ordinary  temperature,  and  in  two 

or  three  days  has  formed  a  slimy  layer 

•of  greyish-yellow  colour,  which  rapidly  '      \>V  / 

spreads  over  the  potato.     On  all  the  ^  6  '//      ^"* 

media  the  growth  has  a  distinctly  foetid  ^     05s  '        (  /^ 

odour.     A  growth  in  peptone  solution         /A    ^^NT   >y^      s     /*    x^' 

fails  to  give  the  cholera-red  reaction     J^    "">?#)$      fy     C 

at  the  end  of  twenty-four  hours,  though    jyv  e\t\^    \f\</        v  ^ 

later  a  faint  reaction  may  appear.     As    '$      ^       *"  N      \T\f-JX      <«'£="    i\ 

stated  above,  Koch  succeeded  in  pro-    ^    ^  .Js^-3^' f  0^T"   ; 

ducing,  by  this  organism,  an  intestinal     '  ^^vfi^^  —^'"^'//i* 

affection  in  guinea-pigs  after  neutralis-  -^-^X     -  ,-£- 

ing  the  stomach  contents  and  paralys-  ^jAjL  s       •' 

ing  the  intestine  with  opium.     This  v*»j,v         S\  \  N"/  '£ 

occurs  in  a  small  proportion  of  the  „  ^  > 

animals    experimented    on,    and    the  v  v>  l^" 

contents  of  the  intestine,  unlike  what       FrG-  J45-  —  Finkler  and  Prior's  spirillum; 

was  found  in  the  case  of  the  cholera   from  an  agar  culture  of  twenty-four  hours' 

growth. 

organism,  were  turbid  in  appearance,       Stained  with  carbol-fuchsin.     x  1000. 
and   had    a    markedly   foetid    odour. 

When  tested  by  intraperitoneal  injection,  its  effects  are  somewhat  of  the  same 
nature  as  those  of  the  cholera  organism,  but  its  virulence  is  of  a  much  lower 
order. 

An  organism  cultivated  by  Miller  ("  Miller's  spirillum  ")  from  the  cavity 
of  a  decayed  tooth  in  a  human  subject  is  almost  certainly  the  same  organism 
as  Finkler  and  Prior's  spirillum. 

Deneke's  Spirillum.  —  This  organism  was  obtained  from  old  cheese,  and 
is  also  known  as  the  spirillum  tyrogennm.  It  closely  resembles  Koch's  spi- 
rillum in  microscopic  appearances,  though  it  is  rather  thinner  and  smaller. 
Its  growth  in  gelatin  is  also  somewhat  similar,  but  liquefaction  proceeds  more 
rapidly,  and  the  bell-shaped  depression  on  the  surface  is  larger  and  shallower, 
whilst  the  growth  has  a  more  distinctly  yellowish  tint.  The  colonies  in  plates 
also  show  points  of  resemblance,  though  the  youngest  colonies  are  rather 
smoother  and  more  regular  on  the  surface,  and  liquefaction  occurs  more 
rapidly  than  in  the  case  of  the  cholera  organism.  The  colonies  have,  on 
naked-eye  examination,  a  distinctly  yellowish  colour.  The  organism  does  not 
give  the  cholera-red  reaction,  and  on  potato  it  forms  a  thin  yellowish  layer 
when  incubated  above  30°  C.  When  tested  by  intraperitoneal  injection  and 
by  other  methods  it  is  found  to  possess  very  feeble,  or  almost  no,  pathogenic 
properties.  Koch  found  that  this  organism,  when  administered  through  the 
stomach  in  the  same  way  as  the  cholera  organism,  produced  a  fatal  result  in 
three  cases  out  of  fifteen.  Deneke's  spirillum  is  usually  regarded  as  a  com- 
paratively harmless  saprophyte. 


CHAPTER   XIX. 

INFLUENZA,    PLAGUE,     RELAPSING     FEVER,     MALTA     FEVER, 
YELLOW   FEVER. 

INFLUENZA. 

THE  first  account  of  the  organism  now  known  as  the  influenza 
bacillus  was  published  simultaneously  by  Pfeiffer,  Kitasato,  and 
Canon,  in  January,  1892.  The  two  first-mentioned  observers 
found  it  in  the  bronchial  sputum,  and  obtained  pure  cultures, 
and  Canon  observed  it  in  the  blood  in  a  few  cases  of  the  disease. 
It  is,  however,  to  Pfeiffer's  work  that  we  owe  most  of  our 

knowledge  regarding  its  char- 

U  '',:      t  i  acters  and  action.     His  results 

.,',.*  .  have  been  amply  confirmed  by 

,   *    -      ',.     _      ^,*  those  of  others  in  various  epi- 

*  *  ',.       V*  !»£>**  **  demies  of  the  disease,  and  this 

i/i  f     -1'*^^  „  '   "        •  1 1       organism  is  now  generally  ac- 

-  '„  'v/'>  ''*•{''  '.  >  •         '  *  '     cepted    as    the    cause    of    the 

* '     r   '  '      "  •       •   *•>•          . 
•%    .*'  \*  ,  '  ^  -•  \    ,  -     -*•*/•        disease,  although  the  absolute 

*  "-         '•"'..  f  •-*   .^r  proof,  which  would  be  supplied 

*•     ^    *       ^   '  "\'7  by  the  production  of  the  dis- 

.    %  "f        *4  '^    ^'    ,  s»  ease  by  pure  cultures,  is  still 

,  ^  4    f  » >  ,       '  .  wanting. 

-li--%  Microscopical  Characters. — 

FIG  146. -influenza  bacilli  from  a  cui-  j^e  influenza  bacilli  as  seen 

ture  on  blood  agar. 

stained  with  carboi-fuchsin.    x  1000.          in  the  sputum  are  very  minute 

rods  not   exceeding    1.5   /-i  in 

length  and  .3  /JL  in  thickness.  They  are  straight,  with  rounded 
ends,  and  sometimes  stain  more  deeply  at  the  extremities  (Fig. 
146).  The  bacilli  occur  singly  or  form  clumps  by  their  aggre- 
gation, but  do  not  grow  into  chains.  They  show  no  capsule. 
They  take  up  the  basic  aniline  stains  somewhat  feebly,  and  are 
best  stained  by  a  weak  solution  (i  in  10)  of  carboMuchsin  applied 

43° 


CULTIVATION   OF   B.    INFLUENZA.  431 

for  5  to  10  minutes.  They  lose  the  stain  in  Gram's  method. 
They  are  non-motile,  and  do  not  form  spores. 

In  many  cases  of  the  disease,  especially  in  the  early  stages 
of  the  more  acute,  influenza  bacilli  are  present  in  large  numbers 
and  may  be  easily  found.  On  the  other  hand,  it  is  often  diffi- 
cult or  impossible  to  find  them,  even  when  the  symptoms  are 
severe ;  this  may  be  due  to  the  restriction  of  the  organisms  to 
some  part  not  readily  accessible,  or  it  may  be  that  they  actually 
die  out  in  great  part,  while  the  effects  of  their  toxins  persist.  It 
has  also  been  observed  in  the  most  recent  epidemic,  in  which 
the  disease  has  been  less  widespread  and  on  the  whole  less 
severe,  that  the  period  during  which  the  bacilli  have  been  readily 
demonstrable  in  the  secretions  has  been  on  the  average  shorter 
than  in  the  previous  epidemics. 

Cultivation.  —  The  best  medium  for  the  growth  of  the  influ- 
enza bacillus  is  blood  agar  (see  p.  42),  which  was  introduced  by 
Pfeiffer  for  this  purpose.  He  obtained  growths  of  the  bacilli 
on  agar  which  had  been  smeared  with  influenza  sputum,  but  he 
failed  to  get  any  ^w^-cultures  on  the  agar  media  or  on  serum. 
The  growth  in  the  first  cultures  he  considered  to  be  probably 
due  to  the  presence  of  certain  organic  substances  in  the  sputum, 
and  accordingly  he  tried  the  expedient  of  smearing  the  agar 
with  drops  of  blood  before  making  the  inoculations.  In  this 
way  he  completely  succeeded  in  attaining  his  object.  The  blood 
of  the  lower  animals  is  suitable,  as  well  as  human  blood.  The 
colonies  of  the  influenza  bacilli  on  blood  agar,  incubated  at 
37°  C.,  appear  within  twenty-four  hours,  in  the  form  of  minute 
circular  dots  almost  completely  transparent.  When  numerous, 
the  colonies  are  scarcely  visible  to  the  naked  eye,  but  when 
sparsely  arranged  they  may  reach  the  size  of  a  small  pin's  head. 
This  size  is  generally  reached  on  the  second  day.  The  bacilli 
die  out  somewhat  quickly  in  cultures,  and  in  order  to  keep  them 
alive,  sub-cultures  should  be  made  every  four  or  five  days.  By 
this  method  the  cultures  may  be  maintained  for  an  indefinite 
period.  They  also  grow  well  on  agar  smeared  with  a  solution 
of  haemoglobin ;  growth  on  the  ordinary  agar  media  is  slight 
and  somewhat  uncertain.  A  very  small  amount  of  growth  takes 
place  in  bouillon,  but  it  is  more  marked  when  a  little  fresh  blood 
is  added.  The  growth  forms  a  thin  whitish  deposit  at  the  bot- 
tom of  the  flask.  The  limits  of  growth  are  from  25°  to  42°  C., 


432  INFLUENZA. 

the  optimum  temperature  being  that  of  the  body.  The  influenza 
bacillus  is  a  strictly  aerobic  organism. 

The  powers  of  resistance  of  this  organism  are  of  a  low  order. 
Pfeiffer  found  that  dried  cultures  kept  at  the  ordinary  tempera- 
ture were  usually  dead  in  twenty  hours,  and  that  if  sputum  were 
kept  in  a  dry  condition  for  two  days,  all  the  influenza  bacilli 
were  dead,  or  rather  cultures  could  be  no  longer  obtained. 
Their  duration  of  life  in  ordinary  water  is  also  short,  the  bacilli 
usually  being  dead  within  two  days.  From  these  experiments 
Pfeiffer  concludes  that  outside  the  body  in  ordinary  conditions 
they  cannot  multiply,  and  can  remain  alive  only  for  a  short  time. 
The  mode  of  infection  in  the  disease  he  accordingly  considers 
to  be  chiefly  by  means  of  mucus,  etc. 

Distribution  in  the  Body.  —  The  bacilli  are  found  chiefly  in 
the  respiratory  passages  in  influenza.  They  may  be  present  in 
large  numbers  in  the  nasal  secretion,  generally  mixed  with  a 
considerable  number  of  other  organisms,  but  it  is  in  the  small 
masses  of  greenish-yellow  sputum  from  the  bronchi  that  they 
occur  in  largest  numbers,  and  in  many  cases  almost  in  a  state  of 
purity.  They  occur  in  clumps  which  may  contain  as  many  as 
100  bacilli,  and  in  the  early  stages  of  the  disease  are  chiefly  lying 
free.  As  the  disease  advances,  they  may  be  found  in  consider- 
able numbers  within  the  leucocytes,  and  towards  the  end  of  the 
disease  a  large  proportion  have  this  position.  It  is  a  matter  of 
considerable  importance,  however,  that  they  may  persist  for 
weeks  after  symptoms  of  the  disease  have  disappeared,  and  may 
still  be  detected  in  the  sputum.  Especially  is  this  the  case  when 
there  is  any  chronic  pulmonary  disease.  They  also  occur  in 
large  numbers  in  the  capillary  bronchitis  and  catarrhal  pneu- 
monia of  influenza,  as  Pfeiffer  showed  by  means  of  sections  of 
the  affected  parts.  In  these  sections  he  found  the  bacilli  lying 
amongst  the  leucocytes  which  filled  the  minute  bronchi,  and 
also  penetrating  between  the  epithelial  cells  and  into  the  super- 
ficial parts  of  the  mucous  membrane.  Their  presence  sets  up  a 
marked  leucocytic  emigration  in  the  peribronchial  tissue,  the 
leucocytes  passing  in  large  numbers  into  the  lumen  of  the  tubes 
and  sometimes  taking  up  the  bacilli.  Other  organisms  also, 
especially  Fraenkel's  pneumococcus,  may  be  concerned  in  the 
pneumonic  conditions  following  influenza. 

In  some  cases  influenza  occurs  in  tubercular  subjects,  or  is 


DISTRIBUTION   IN    THE   BODY.  433 

followed  by  tubercular  affection,  in  which  cases  both  influenza 
and  tubercle  bacilli  may  be  found  in  the  sputum.  In  such  a  con- 
dition the  prognosis  is  very  grave.  Regarding  the  presence  of 
influenza  bacilli  in  the  other  pulmonary  complications  following 
influenza,  much  information  is  still  required.  Occasionally  in 
the  foci  of  suppurative  softening  in  the  lung  the  influenza  bacilli 
have  been  found  in  a  practically  pure  condition.  In  cases  of 
empyema  the  organisms  present  would  appear  to  be  chiefly 
streptococci  and  pneumococci ;  whilst  in  the  gangrenous  condi- 
tions, which  sometimes  occur,  a  great  variety  of  organisms  has 
been  found. 

As  above  stated,  Canon  described  the  bacilli  as  occurring  in 
the  blood  during  life,  and  Pfeiffer,  on  examining  Canon's  prepa- 
rations, admits  that  the  bacilli  shown  resembled  the  influenza 
bacilli.  His  own  observations  on  a  large  series  of  cases  con- 
vinced him  that  the  organism  was  very  rarely  present  in  the 
blood,  that  in  fact  its  occurrence  there  must  be  looked  upon  as 
exceptional.  The  conclusions  of  other  observers  have,  on  the 
whole,  confirmed  this  statement.  The  organism  has  been  regu- 
larly found  in  enormous  numbers  in  the  sputum  in  influenza,  but 
only  occasionally  and  in  small  numbers  in  the  blood.  It  is  prob- 
able that  the  chief  symptoms  in  the  disease  are  due  to  toxins 
absorbed  from  the  respiratory  tract  (vide  infra], 

We  cannot  yet  speak  definitely  with  regard  to  the  relation  of 
the  bacillus  in  other  complications  in  influenza.  Pfeiffer  found 
it  in  inflammation  of  the  middle  ear,  but  in  a  case  of  meningitis 
following  influenza,  Fraenkel's  diplococcus  was  present.  In  a 
few  cases  of  meningitis,  however,  the  influenza  bacillus  has  been 
found,  sometimes  alone,  sometimes  along  with  pyogenic  cocci 
(Pf uhl  and  Walter,  Cornil  and  Durante) ;  Pfuhl  considers  that 
in  these  the  path  of  infection  is  usually  a  direct  one  through  the 
roof  of  the  nasal  cavity.  This  observer  also  found  post  mortem, 
in  a  rapidly  fatal  case  with  profound  general  symptoms,  influenza 
bacilli  in  various  organs,  both  within  and  outside  of  the  vessels. 
In  a  few  cases  also  the  bacilli  have  been  found  in  the  brain  and 
its  membranes,  with  little  tissue  change  in  the  parts  around. 

Experimental  Inoculation.  —  There  is  no  satisfactory  evidence 
that  any  of  the  lower  animals  suffer  from  influenza  in  natural 
conditions,  and  accordingly  we  cannot  look  for  very  definite 
results  from  experimental  inoculation.  Pfeiffer,  by  injecting 


434 


INFLUENZA. 


living  cultures  of  the  organism  into  the  lungs  of  monkeys,  in 
three  cases  produced  a  condition  of  fever  of  a  remittent  type. 
Somewhat  similar  results  were  obtained  in  one  animal  by  smearing 
the  uninjured  mucous  membrane  of  the  nose  with  a  pure  culture. 
The  fever  appeared  about  twenty-four  hours  after  the  injection, 
and  lasted  for  from  three  to  five  days.  In  another  case  in  which 
large  quantities  of  the  bacilli  were  injected  into  the  trachea, 
marked  prostration  and  high  temperature  occurred,  death  follow- 
ing in  twenty-four  hours.  There  was,  however,  little  evidence  that 
the  bacilli  had  undergone  multiplication,  the  symptoms  being 
apparently  produced  by  their  toxins.  In  the  case  of  rabbits, 
intravenous  injection  of  living  cultures  produces  dyspnoea,  mus- 
cular weakness,  and  slight  rise  of  temperature,  but  the  bacilli 
rapidly  disappear  in  the  body,  and  exactly  similar  symptoms 
are  produced  by  injection  of  cultures  killed  by  the  vapour  of 
chloroform.  Pfeiffer,  therefore,  came  to  the  conclusion  that  the 
influenza  bacilli  contain  toxic  substances  which  can  produce  in 
animals  some  of  the  substances  of  the  disease,  but  that  animals 
are  not  liable  to  infection,  the  bacilli  not  having  power  of  multi- 
plying to  any  extent  in  their  tissues. 

Cantani  in  a  recent  work  succeeded  in  producing  infection  to 
some  extent  in  rabbits,  by  injecting  the  bacilli  directly  into  the 
anterior  portion  of  the  brain.  In  these  experiments  the  organisms 
spread  to  the  ventricles,  and  then  through  the  spinal  cord  by 
means  of  the  central  canal,  afterwards  infecting  the  substance  of 
the  cord.  An  acute  encephalitis  was  thus  produced,  and  some- 
times a  purulent  condition  in  the  lateral  ventricles.  The  bacilli, 
were,  however,  never  found  in  the  blood  or  in  other  organs.  The 
symptoms  produced  were  great  dyspnoea,  cardiac  weakness,  and 
also  a  paralytic  condition  which  appeared  first  in  the  posterior 
limbs,  and  then  spread  to  the  rest  of  the  body.  The  temperature 
was  at  first  elevated,  but  before  death  fell  below  normal.  Similar 
symptoms  were  also  produced  by  injection  of  dead  cultures, 
though  in  this  case  the  dose  required  to  be  five  or  six  times 
larger.  Cantani  therefore  concludes  that  the  brain  substance  is 
the  most  suitable  nidus  for  their  growth,  but  agrees  with  Pfeiffer 
in  believing  that  the  chief  symptoms  are  produced  by  toxins 
resident  in  the  bodies  of  the  bacilli.  He  made  control  experi- 
ments by  injecting  other  organisms,  and  also  by  injecting  inert 
substances  into  the  cerebral  tissue. 


PLAGUE. 


435 


The  evidence,  accordingly,  that  the  influenza  bacillus  is  the 
cause  of  the  disease  rests  chiefly  on  the  well-established  fact 
that  it  is  always  present  in  the  secretions  of  the  respiratory 
tract  in  true  cases  of  influenza,  and  that  it  is  an  organism  which 
has  not  been  found  in  any  other  condition.  Moreover,  it  is  an 
organism  which  is  practically  restricted  by  its  conditions  of 
growth  to  the  animal  body.  A  certain  amount  of  confirmatory 
evidence  has  been  supplied  by  the  results  of  experiment. 

Methods  of  Examination. — (a)  Microscopic.  —  A  portion  of 
the  greenish-yellow  purulent  material  which  often  occurs  in 
little  round  masses  in  the  sputum  should  be  selected,  and  film 
preparations  should  be  made  in  the  usual  way.  Films  are  best 
stained  by  Ziehl-Neelsen  carbol-fuchsm  diluted  with  ten  parts 
of  water,  the  films  being  stained  for  ten  minutes  at  least.  In 
sections  of  the  tissues,  such  as  the  lungs,  the  bacilli  are  best 
brought  out,  according  to  Pfeiffer,  by  staining  with  the  same 
solution  as  above  for  half  an  hour.  The  sections  are  then 
placed  in  alcohol  containing  a  few  drops  of  acetic  acid,  in 
which  they  are  dehydrated  and  slightly  decolorised  at  the  same 
time.  They  should  be  allowed  to  remain  till  they  have  a  mod- 
erately light  colour,  the  time  varying  according  to  their  appear- 
ance. They  are  then  washed  in  pure  alcohol,  cleared  in  xylol, 
and  afterwards  mounted  in  balsam. 

(b)  Cultures.  —  A  suitable  portion  of  the  greenish-yellow 
material  having  been  selected  from  the  sputum,  it  should  be 
washed  well  in  several  changes  of  sterilised  water.  A  portion 
should  then  be  taken  on  a  platinum  needle,  and  successive 
strokes  made  on  the  surface  of  blood-agar  tubes.  The  tubes 
should  then  be  incubated  at  37°  C.,  when  the  transparent 
colonies  of  the  influenza  bacillus  will  appear,  usually  within 
twenty-four  hours.  These  should  give  a  negative  result  on 
inoculation  on  ordinary  agar  media. 

PLAGUE.1 

The  bacillus  of  oriental  plague  or  bubonic  pest  was  discovered 
independently  by  Kitasato  and  Yersin  during  the  epidemic  at 
Hong  Kong  in  1894.  The  results  of  their  investigations,  which 
were  published  almost  at  the  same  time,  agree  in  most  of  the 

1  In  revising  this  subject  we  have  made  extensive  use  of  the  Report  of  the  Indian 
Plague  Commission,  1901. 


436 


PLAGUE. 


important  points.     They  cultivated  the  same  organism  from  a 

large  numberof 

^'-'5     *H/Jf  *i'dl^  cases  of  plague, 

i'lHlfelVltl  fjr^/  and  reproduced 

*<    ^  i?J5&  MS,  the   disease   in 

*WC       '^^^4  susceptible  ani- 

mals by  inocu- 
lation of  pure 
cultures.  It  is 
to-  be  noted 
that  during  an 
epidemic  of 
plague,  some- 
times even  pre- 
ceding it,  a  high 
mortality  has 
been  observed 
among  certain 
animals,  espe- 

FlG.  147.  — Film  preparation  from  a  plague  bubo  showing  claUv  ra-tS  and 

enormous  numbers  of  bacilli,  most  of  which  show  well-marked  bi-  mice,  and  that 

polar  staining.  ,-  ,  ,  , . 

Stained  with  weak  gentian-violet.     X  1000. 

of  these  ani- 
mals found  dead  in  the  plague-stricken  district,  the  same  bacillus 
was  obtained  by  Kitasato  and  ..»- 

also  by  Yersin.  r.  «\     v  /  •  »  ,    '- 

Bacillus  of  Plague.  —  Micro-         .v  -    '  v   y    ""**•*  ^  ~\~*\ 
scopical  Characters.  —  As  seen       '   .'    \       '4\*\  -    '% 
in  the  affected  glands  or  buboes  - -,%  1        *       \  !  •   <••  >    ^ 

in    this     disease,     the     bacilli         •  . -^'  ^«  ^    \*\ •'  ^"' 
are  small  oval  rods,  somewhat        *     v>   »''*'{;v  *  -^ 
shorter  than  the  typhoid  bacil-     .     ,\  >  x     +•'•-.'*' ,  \  '/  '  ^'      '* 
lus,     and      about     the     same  : \  *  '  %%% 

thickness  (Fig.  148),  though 
considerable  variations  in  size 
occur.  They  have  rounded 
ends,  and  in  stained  prepara- 


•'/      ^  -  v      .  \ 

xx--~\ 
.*<^-     ...  - 

FlG.  I48._  Bacillus  of  plague  from  a 


tions  a  portion  in  the  middle  of   y°uns  culture  on  agar. 

Stained  with  weak  carbol-fuchsin.   Xiooo. 

the  bacillus  is  often  left  uncov- 

ered, giving  the  so-called  "  polar  staining."     In  films  from  the 


BACILLUS   PESTIS.  437 

tissues  they  are  found  scattered  amongst  the  cells,  for  the  most 

part  lying  singly,  though  pairs  are  also  seen.     On  the  other 

hand,  in  cultures  in  fluids,  e.g. 

bouillon,  they  grow  chiefly  in 

chains,  sometimes  of  consider-  \        \ 

able    length,  the  form   known  ^  x 

as    a   streptobacillus    resulting  \  / 

(Fig.  149).     In  young  agar  cul-         ^N         f*t         \ 

tures  the  bacilli  show  greater 

variation    in    size,    and    polar  % 

staining  is  less  marked  than  in  \        t  / 

****  *^  / 

the  tissues  :    sometimes  forms  *X 

of  considerable  length  are  pres-  ^s          \  / 

ent.     After  a  time  involution 
forms  appear,  especially  when 

r  r  •  T49'  —  Bacillus  of  plague  in  chains, 

the  Surface  Of    the  agar  IS  dry;     showing,  polar  staining.     From  a  young  cul- 

but  the  formation  of   these  is   ^  in  bouillon. 

Stained  with  thionm-blue.     x  1000. 

much    more    rapid    and    more 

marked  when  2-5  per  cent  of  sodium  chloride  is  added  to  the 
medium,  constituting  the  so-called  "salt  agar"  (Hankin  and 
Leumann).  On  this  medium,  especially  with  the  higher  per- 

centage, the   involution   forms 

^*  "'  •;*•-"      •    C     .  assume  a  great  size  and  a  strik- 

ing  variety    of    shapes,    large 

0+  •          ^#  *****   •  globular,     oval,     or     pyriform 

*^.T  bodies    resulting    (Fig.     150); 

with  about  2  per  cent  sodium 
chloride,,     after      twenty-four 
hours'    incubation,    the    most 
*4  I  v      striking   feature   is   a    general 

£*  enlargement  of  all  the  bacilli. 

Sometimes  in  the  tissues  they 
*      „*      *  *         /  J 

are  seen  to  be  surrounded  by 
an   unstained   capsule,   though 

FIG.    150.  —  Culture   of    the   bacillus   of      ,  ,  •  .     -, 

plague  on  4  per  cent  salt  agar,  showing  in-    thls  appearance  is  by  no  means 

volution  forms  of  great  variety  of  size  and     common.       They    do    not    form 

stained  with  carboi-thionin-biue.   xiooo.    spores.   Gordon,  who  has  found 

that  they  possess  flagella  which, 

however,  stain  with  difficulty,  states  that  they  are  motile.  Most 
observers,  however,  and  with  these  we  agree,  have  failed  to 


438  PLAGUE. 

find   evidence  of  true  motility.      They  stain  readily  with  the 
basic  aniline  stains,  but  are  decolorised  by  Gram's  method. 

Cultivation.  —  From  the  affected  glands,  etc.,  the  bacillus 
can  readily  be  cultivated  on  the  ordinary  media.  It  grows  best 
at  the  temperature  of  the  body,  though  growth  occurs  as  low  as 
1 8°  C.  On  agar  and  on  blood  serum  the  colonies  are  circular 
discs  of  somewhat  transparent  appearance  and  smooth,  shining 
surface.  When  examined  with  a  lens,  their  borders  appear 
slightly  wavy.  In  stroke-cultures  on  agar  there  forms  a  con- 
tinuous line  of  growth  with  the  same  appearance,  showing  partly 
separated  colonies  at  its  margins.  When  agar  cultures  are  kept 
at  the  room  temperature,  some  of  the  colonies  show  a  more 
luxuriant  growth  with  more  opaque  appearance  than  the  rest  of 
the  growth,  the  appearance  in  fact  being  often  such  as  to  sug- 
gest the  presence  of  impurities  in  the  cultures.  In  stab-cultures 
in  peptone  gelatin,  growth  takes  place  along  the  needle  track  as 
a  white  line,  composed  of  small  spherical  colonies.  On  the  sur- 
face of  the  gelatin  a  thin,  semi-transparent  layer  may  be  formed, 
which  is  usually  restricted  to  the  region  of  puncture,  though 
sometimes  it  may  spread  to  the  wall  of  the  tube ;  sometimes, 
however,  there  is  practically  no  surface  growth.  There  is  no 
liquefaction  of  the  medium.  In  gelatin  plates  the  superficial 
colonies  develop  first  and  form  slightly  raised  semi-transparent 
discs  with  somewhat  crenated  margins;  the  deeper  colonies  are 
smaller  and  of  spherical  shape  with  smooth  outline.  In  bouillon 
the  growth  usually  forms  a  slightly  granular  or  powdery  deposit 
at  the  foot  and  sides  of  the  flask,  somewhat  resembling  that  of 
a  streptococcus.  If  oil  or  melted  butter  is  added  to  the  bouillon 
so  that  drops  float  on  the  surface,  then  a  striking  mode  of  growth 
may  result,  to  which  the  term  "stalactite"  has  been  applied. 
This  consists  in  the  growth  starting  from  the  under  surface 
of  the  fat  globules  and  extending  downwards  in  the  form  of 
pendulous,  string-like  masses.  These  masses  are  exceedingly 
delicate,  and  readily  break  off  on  the  slightest  shaking  of  the 
flask ;  accordingly  during  their  formation  the  culture  must  be 
kept  absolutely  at  rest.  This  manner  of  growth  constitutes 
an  important  but  not  absolutely  specific  character  of  the 
organism;  unfortunately  it  is  not  supplied  by  all  races  of 
the  organism,  and  varies  from  time  to  time  with  the  same 
race.  The  organism  flourishes  best  in  an  abundant  supply  of 


DISTRIBUTION   OF   THE    BACILLI. 


439 


oxgyen ;  in  strictly  anaerobic  conditions  almost  no  growth  takes 
place. 

The  organism  in  its  powers  of  resistance  corresponds  with 
other  spore-free  bacilli,  and  is  readily  killed  by  heat,  an  exposure 
for  an  hour  at  58°  C.  being  fatal.  On  the  other  hand,  it  has 
remarkable  powers  of  resistance  against  cold ;  it  has  been  ex- 
posed to  a  temperature  several  degrees  below  freezing-point 
without  being  killed.  Experiments  on  the  effects  of  drying 
have  given  somewhat  diverse  results,  but  as  a  rule  the  organism 
has  been  found  to  be  dead  after  being  dried  for  from  six  to  eight 
days,  though  sometimes  it  has  survived  the  process  for  a  longer 
period ;  exposure  to  direct  sunlight  for  three  or  four  hours 
kills  it.  When 
cultivated  out- 
side  the  body 
the  organism 
often  loses  its 
virulence,  but 
some  races  re- 
main virulent 
in  culture  for 
a  long  period 
of  time. 

Anatomical 
Changes  and 
Distribution  of 
Bacilli.  —  The 
disease  occurs 
in  several 
forms,  the  bu- 
bonic and  the 


FIG.  151.  —  Section   of  a  human  lymphatic  gland  in  plague, 
showing  the  injection  of  the  lymph  paths  and  sinuses  with  masses 


pulmonary    be-      °^  PlaSue  bacilli  —  seen  as  black  areas. 

Stained  with  carbol-thionin-blue.     X  50. 

ing     the     best 

recognised ;  to  these  may  be  added  the  septiccemic.  The  most 
striking  feature  in  the  bubonic  form  is  the  affection  of  the 
lymphatic  glands,  which  undergo  intense  inflammatory  swell- 
ing, attended  with  haemorrhage,  and  generally  ending  in  a 
greater  or  less  degree  of  necrotic  softening  if  the  patient  lives 
long  enough.  The  connective  tissue  around  the  glands  is 
similarly  affected.  The  bubo  is  thus  usually  formed  by  a  col- 


440  PLAGUE. 

lection  of  enlarged  glands  fused  by  the  inflammatory  swelling. 
True  suppuration  is  rare.  Usually  one  group  of  glands  is 
affected  first,  constituting  the  primary  bubo- — in  the  great 
majority  the  inguinal  or  the  axillary  glands  —  and  afterwards 
other  groups  may  become  involved,  though  to  a  much  less 
extent.  Along  with  these  changes  there  is  great  swelling  of 
the  spleen,  and  often  intense  cloudy  swelling  of  the  cells  of  the 
kidneys,  liver,  and  other  organs.  There  may  also  occur  second- 
ary areas  of  haemorrhage  and  the  necrosis,  chiefly  in  the  lungs, 
liver,  and  spleen.  The  bacilli  occur  in  enormous  numbers  in 
the  swollen  glands,  being  often  so  numerous  that  a  film  prepa- 
ration made  from  a  scraping  almost  resembles  a  pure  culture 
(Fig.  147).  In  sections  of  the  glands  in  the  earlier  stages  the 
bacilli  are  found  to  form  dense  masses  in  the  lymph  paths  and 
sinuses  (Fig.  151),  often  forming  an  injection  of  them  ;  they  may 
also  be  seen  growing  as  a  fine  reticulum  between  the  cells  of 
the  lymphoid  tissue.  At  a  later  period,  when  disorganisation 
of  the  gland  has  occurred,  they  become  irregularly  mixed  with 
the  cellular  elements.  Later  still  they  gradually  disappear,  and 
when  necrosis  is  well  advanced  it  may  be  impossible  to  find  any 
—  a  point  of  importance  in  connection  with  diagnosis.  In  the 
spleen  they  may  be  very  numerous  or  they  may  be  scanty, 
according  to  the  amount  of  blood  infection  which  has  occurred ; 
in  the  secondary  lesions  mentioned  they  are  often  abundant. 
In  the  pulmonary  form  the  lesion  is  the  well-recognised  "plague 
pneumonia."  This  is  of  broncho-pneumonic  type,  though  large 
areas  may  be  formed  by  confluence  of  the  consolidated  patches, 
and  the  inflammatory  process  is  attended  usually  by  much 
haemorrhage ;  the  bronchial  glands  show  inflammatory  swelling. 
Clinically  there  is  usually  a  fairly  abundant  frothy  sputum  often 
tinted  with  blood,  and  in  it  the  bacilli,  may  be  found  in  large 
numbers.  Sometimes,  however,  cough  and  expectoration  may 
be  absent.  The  disease  "in  this  form  is  said  to  be  invariably 
fatal.  In  the  septiccemic  form  proper  there  is  no  primary  bubo 
discoverable,  though  there  is  almost  always  general  enlargement 
of  lymphatic  glands ;  here  also  the  disease  is  of  specially  grave 
character.  A  bubonic  case  may,  however,  terminate  with  sep- 
ticaemia ;  in  fact  all  intermediate  forms  occur.  An  intestinal 
form  with  extensive  affection  of  the  mesenteric  glands  has  been 
described,  but  it  is  exceedingly  rare  —  so  much  so  that  many 


EXPERIMENTAL   INOCULATION.  441 

observers  with  extensive  experience  have  doubted  its  occurrence. 
In  the  various  forms  of  the  disease  the  bacilli  occur  also  in  the 
blood,  in  which  they  may  be  found  during  life  by  microscopic 
examination,  chiefly,  however,  just  before  death  in  very  severe 
and  rapidly  fatal  cases.  In  most  cases,  however,  they  cannot  be 
detected  in  the  blood  by  this  means,  though  in  some  of  these 
they  may  be  obtained  by  means  of  cultures. 

The  above  types  of  the  disease  are  usually  classified  together 
under  the  heading  pestis  major,  but  there  also  occur  mild  forms 
to  which  the  term  pestis  minor  is  applied.  In  these  latter  there 
may  be  a  moderate  degree  of  swelling  of  a  group  of  glands, 
attended  with  some  pyrexia  and  general  malaise,  or  there  may 
be  little  more  than  slight  discomfort.  Between  such  and  the 
graver  types,  cases  of  all  degrees  of  severity  are  met  with. 

Experimental  Inoculation.  —  Mice,  guinea-pigs,  rats,  and 
rabbits  are  susceptible  to  inoculation,  the  two  former  being 
on  the  whole  most  suitable  for  experimental  purposes.  After 
subcutaneous  injection  there 
occurs  a  local  inflammatory 
oedema,  which  is  followed 
by  inflammatory  swelling  of 
the  corresponding  lymphatic 
glands,  and  thereafter  by  a 
general  infection.  The  le- 
sions in  the  lymphatic  glands 
correspond  in  their  main  char- 
acters with  those  in  the  human 
subject,  although  usually  at  the 
time  of  death  they  have  not 
reached  a  stage  so  advanced. 

By  this  method  Of  inoculation  Flfa  ^—Fihn  preparation  of  spleen  of 

•;  ^  rat  alter  inoculation  with  bacillus  of  plague, 

mice   Usually  die  in    1—3   days,     showing  numerous  bacilli,  most  of  which  are 


guinea-pigs  and  rats  in  2-5 
days,  and  rabbits  in  4-7  days. 
Post  mortem  the  chief  changes,  in  addition  to  the  glandular 
enlargement,  being  congestion  of  internal  organs,  sometimes 
with  haemorrhages,  and  enlargement  of  the  spleen ;  the  bacilli 
are  numerous  in  the  lymphatic  glands  and  usually  in  the  spleen 
(Fig.  152),  and  also,  though  in  somewhat  less  degree,  through- 
out the  blood.  Infection  can  also  be  produced  by  smearing  the 


442 


PLAGUE. 


material  on  the  conjunctiva  or  mucous  membrane  of  the  nose, 
and  this  method  of  inoculation  has  been  successfully  applied 
in  cases  where  the  plague  bacilli  are  present  along  with  other 
virulent  organisms,  e.g.  in  sputum  along  with  pneumococci. 
Rats  and  mice  can  also  be  infected  by  feeding  either  with 
pure  cultures  or  with  pieces  of  organs  from  cases  of  the  disease, 
though  in  this  case  infection  probably  takes  place  through  the 
mucous  membrane  of  the  mouth  and  adjacent  parts,  and  only 
to  a  limited  extent,  if  at  all,  by  the  alimentary  canal.  Monkeys 
also  are  highly  susceptible  to  infection,  and  it  has  been  shown 
fn  the  case  of  these  animals  that,  when  inoculation  is  made  on 
the  skin  surface,  for  example,  by  means  of  a  spine  charged  with 
the  VJacillus,  the  glands  in  relation  to  the  part  may  show  the 
characteristic  lesion  and  a  fatal  result  may  follow  without  there 
being  any  noticeable  lesion  at  the  primary  seat.  This  fact  throws 
important  light  on  infection  by  the  skin  in  the  human  subject. 
The  disease  may  also  extensively  affect  monkeys  by  natural 
means  during  an  epidemic. 

Paths  and  Mode  of  Infection.  —  It  is  now  well  established  that 
in  the  great  majority  of  cases  plague  bacilli  enter  the  system  by 
the  skin  surface  through  small  wounds,  cracks,  abrasions,  etc., 
and  that  there  is  usually  no  reaction  at  the  site  of  entrance. 
This  last  fact  is  in  accordance  with  what  has  been  stated  above 
with  regard  to  experiments  on  monkeys.  The  path  of  infection 
is  shown  by  the  primary  buboes,  which  are  usually  in  the  glands 
through  which  the  skin  is  drained,  those  in  the  groin  being  the 
commonest  site.  Absolute  proof  of  infection  by  the  skin  is  sup- 
plied by  several  cases  in  which  the  disease  has  been  acquired 
at  post-mortem  examinations,  the  lesions  of  the  skin  surface 
being  in  the  majority  of  these  of  trifling  nature;  in  only  two 
was  there  local  reaction  at  the  site  of  inoculation.  In  most  of 
these  cases  the  period  of  incubation  has  been  from  two  to  three 
days ;  under  natural  conditions  of  infection  the  Indian  Commis- 
sion places  the  average  period  within  five  days.  In  a  small 
proportion  of  cases  infection  takes  place  through  the  mucous 
membrane  of  the  nose  and  mouth,  and  exceptionally  of  other 
parts ;  it  is  still  considered  doubtful  whether  the  alimentary 
canal  is  a  path  of  infection.  In  primary  plague  pneumonia, 
from  a  consideration  of  the  anatomical  changes  and  the  clinical 
facts,  the  disease  may  be  said  to  be  produced  by  the  direct  pas- 


PATHS  AND  MODES  OP^  INFECTION. 


443 


.sage  of  the  bacilli  into  the  respiratory  passages.  Nevertheless 
there  must  be  certain  factors,  still  imperfectly  understood,  which 
determine  the  incidence  of  this  form ;  as  in  some  epidemics  of 
the  highest  virulence  plague  pneumonia  has  been  practically 
absent,  though  opportunities  for  infection  by  inhalation  must 
have  been  present.  On  the  other  hand,  a  case  of  plague  pneu- 
monia is  of  great  infectivity  in  producing  other  cases  of  plague 
pneumonia. 

With  regard  to  the  mode  of  infection  it  may  be  stated,  in  the 
first  place,  that  if  we  except 'plague  pneumonia  and  those  rare 
conditions  in  which  true  plague  eruptions  are  present,  direct 
infection  from  patient  to  patient  is  relatively  uncommon.  This 
is  in  accordance  with  the  fact  that  in  bubonic  plague  the  bacilli 
are  not  discharged  from  the  unbroken  surface  of  the  body,  and 
are  only  present  in  the  secretions  in  severe  cases.  In  the  great 
majority  contagion  is  of  indirect  character,  through  the  medium 
of  soiled  clothes,  f omites  —  in  fact,  contaminated  articles  gener- 
ally ;  thus  rooms  and  houses  come  to  be  "  sources  of  infection." 
In  addition  to  cases  of  the  disease  in  the  human  subject,  the 
affection  in  animals,  especially  in  rats,  plays  an  important  part 
in  the  spread  of  epidemics.  The  disease  in  these  animals  has, 
in  fact,  been  the  means  of  rapidly  distributing  infection  over 
wide  areas  of  a  town  or  district.  This  has  been  abundantly 
proved  in  the  case  of  Bombay,  where  observations  have  shown 
that  the  migration  of  plague-infected  rats  to  quarters  compara- 
tively free  from  the  disease  has  been  followed  by  extensive  out- 
breaks in  these  placesv  On  the  other  hand,  there  is  no  doubt 
that  plague  in  a  very  widespread  and  virulent  form  may  occur 
without  the  agency  of  these  animals. 

The  view  that  fleas  and  other  insects  play  a  part  in  the  spread 
of  plague  has  obtained  pretty  wide  acceptance,  but  recent  exami- 
nation has  proved  that  most  of  the  data  on  which  this  is  based 
are  of  unsatisfactory  nature.  Yersin  found  the  plague  bacillus 
in  the  dead  bodies  of  these  insects,  and  Simond  brought  forward 
the  results  of  experiments  which  appeared  to  show  that  the  fleas 
infecting  a  plague-stricken  rat  left  the  animal  when  it  died,  and 
might  produce  plague  in  a  healthy  animal.  Experiments  per- 
formed by  others  have,  however,  given  negative  results,  and  the 
finding  of  the  Indian  Commission  is  that  suctorial  insects  are 
practically  of  no  importance  as  transmitters  of  infection. 


444  PLAGUE. 

From  the  facts  stated  with  regard  to  the  powers  of  compara- 
tively rapid  multiplication  of  the  bacillus,  its  wide  dissemination 
by  affected  rats,  human  excreta,  etc.,  it  may  be  understood  how 
extensively  the  soil  and  dwellings  may  become  infected,  and 
how  difficult  it  may  be  to  arrest  the  ravages  of  the  disease. 
How  important  a  part  such  infection  of  a  locality  plays  is  strik- 
ingly shown  by  the  rapid  fall  in  the  number  of  cases  when  the 
people  go  into  tents. 

Toxins,  Immunity,  etc.  —  As  is  the  case  with  most  organisms 
which  extensively  invade  the  tissues,  the  toxins  in  plague  cultures 
are  chiefly  contained  in  the  bodies  of  the  bacteria.  Injection  of 
dead  cultures  in  animals  produces  distinctly  toxic  effects ;  post- 
mortem,  haemorrhage  in  the  mucous  membrane  of  the  stomach, 
areas  of  necrosis  in  the  liver  and  at  the  site  of  inoculation,  may 
be  present.  The  toxic  substances  are  comparatively  resistant  to 
heat,  being  unaffected  by  an  exposure  to  65°  C.  for  an  hour.  By 
the  injection  of  dead  cultures  in  suitable  doses  a  certain  degree 
of  immunity  against  the  living  virulent  bacilli  is  obtained,  and, 
as  first  shown  by  Yersin,  Calmette,  and  Borrel,  the  serum  of 
such  immunised  animals  confers  a  degree  of  protection  on 
smaller  animals,  such  as  mice.  On  these  facts  the  principles  of 
preventive  inoculation  and  serum  treatment,  presently  to  be 
described,  depend.  It  may  also  be  mentioned  that  the  filtrate 
of  a  plague  culture  possesses  a  very  slight  toxic  action,  and  the 
Indian  Plague  Commission  found  that  such  a  filtrate  has  prac- 
tically no  effect  in  the  direction  of  conferring  immunity. 

I .  Preventive  Inoculation  —  Haffkine[s  Method.  —  To  prepare 
the  preventive  fluid,  cultures  are  made  in  flasks  of  bouillon  with 
drops  of  oil  on  the  surface  (in  India  Haff kine  employed  a  medium 
prepared  by  digesting  goats'  flesh  with  hydrochloric  acid  at 
140°  C.  and  afterwards  neutralising  with  caustic  soda).  In  such 
cultures  stalactite  growths  (vide  snpra)  form,  and  the  flasks  are 
shaken  every  few  days  so  as  to  break  up  the  stalactites  and 
induce  fresh  crops.  The  flasks  are  kept  at  a  temperature  of 
about  25°  C.,  and  growth  is  allowed  to  proceed  for  about  six 
weeks.  At  the  end  of  this  time  sterilisation  is  effected  by  expos- 
ing the  contents  of  the  flasks  to  65°  C.  for  an  hour ;  thereafter 
carbolic  acid  is  added  in  the  proportion  of  .5  per  cent.  The 
contents  are  well  shaken  to  diffuse  thoroughly  the  sediment  in 
the  fluid  and  are  then  distributed  in  small  sterilised  bottles  for 


PREVENTIVE   INOCULATION.  445 

use.  The  preventive  fluid  thus  contains  both  the  dead  bodies 
of  the  bacilli  and  any  toxins  which  may  be  in  solution.  It  is 
administered  by  subcutaneous  injection,  the  dose,  which  varies 
.according  to  the  "strength,"  being  on  an  average  about  7.5  c.c. 
Usually  only  one  injection  is  made,  sometimes  two ;  though  the 
latter  procedure  does  not  appear  to  have  any  advantage.  The 
method  has  been  systematically  tested  by  inoculating  a  certain 
proportion  of  the  inhabitants  of  districts  exposed  to  infection, 
leaving  others  uninoculated,  and  then  observing  the  proportion 
of  cases  of  disease  and  the  mortality  amongst  the  two  classes. 
The  results  of  inoculation,  as  attested  by  the  Indian  Commission, 
have  been  distinctly  satisfactory.  For  although  absolute  protec- 
tion is  not  afforded  by  inoculation,  both  the  proportion  of  cases 
of  plague  and  the  percentage  mortality  amongst  these  cases 
have  been  considerably  smaller  in  the  inoculated  as  compared 
with  the  uninoculated.  Protection  is  not  established  till  some 
days  after  inoculation,  and  probably  lasts  for  a  considerable 
number  of  weeks.  The  Commission  recommend,  however,  the 
employment  of  a  better  method  of  standardisation  (this  being 
roughly  effected  according  to  the  amount  of  suspended  matter 
present),  and  also  more  efficient  methods  for  ensuring  the  free- 
dom of  the  fluid  from  contaminating  organisms  —  improvements 
which  will  no  doubt  be  carried  out. 

2.  Anti-plague  Sera.  —  Of  these  two  have  been  used  as  thera- 
peutic agents,  viz.,  that  of  Yersin  and  that  of  Lustig.  Yersin's 
serum  is  prepared  by  injections  of  increasing  doses  of  plague 
bacilli  into  the  horse.  In  the  early  stages  of  immunisation  dead 
bacilli  are  injected  subcutaneously,  thereafter  into  the  veins, 
and,  finally,  living  bacilli  are  injected  intravenously.  After  a 
suitable  time  blood  is  drawn  off  and  the  serum  is  preserved  in  the 
usual  way.  Of  this  serum  10-20  c.c.  are  used,  and  injections  are 
usually  repeated  on  subsequent  days.  Lustig's  serum  is  pre- 
pared by  injecting  a  horse  with  repeated  and  increasing  doses  of 
a  substance  derived  from  the  bodies  of  plague  bacilli,  probably 
in  great  part  nucleo-proteid.  Masses  of  growth  are  obtained 
from  the  surface  of  agar  cultures,  and  are  broken  up  and  dis- 
solved in  a  i  per  cent  solution  of  caustic  potash.  The  solution 
is  then  made  slightly  acid  by  hydrochloric  acid,  when  a  bulky 
precipitate  forms  ;  this  is  collected  on  a  filter  and  dried.  For  use 
a  weighed  amount  is  dissolved  in  a  weak  solution  of  carbonate 


446  PLAGUE. 

of  soda  and  then  injected.  The  serum  is  obtained  from  the 
animal  in  the  usual  way.  Extensive  observations  with  both  of 
these  sera  show  that  neither  of  them  can  be  considered  a  pow- 
erful remedy  in  cases  of  plague,  though  in  certain  instances 
distinctly  favourable  results  have  been  recorded.  The  Indian 
Commission,  however,  came  to  the  conclusion  "  that,  on  the 
whole,  a  certain  amount  of  advantage  accrued  to  the  patients 
both  in  case  of  those  injected  with  Yersin's  serum  and  of  those 
injected  with  Lustig's  serum."  It  may  also  be  mentioned  that 
the  Commission  found,  as  the  results  of  experiments,  that 
Yersin's  serum  modified  favourably  the  course  of  the  disease  in 
animals,  whereas  Lustig's  serum  had  no  such  effect. 

3.  Serum  Diagnosis.  —  Specific  agglutinins  may  appear  in 
the  blood  of  patients  suffering  from  plague,  as  also  they  do  in  the 
case  of  animals  immunised  against  the  plague  bacillus.  It  is  to 
be  noted,  however,  that  in  clinical  cases  the  reaction  is  not  in- 
variably present,  the  potency  of  the  serum  is  not  of  high  order,, 
and  the  carrying  out  of  the  test  is  complicated  by  the  natural 
tendency  of  the  bacilli  to  cohere  in  clumps.  For  the  last  reason 
the  macroscopic  (sedimentation)  method  is  to  be  preferred  to  the 
microscopic  (p.  109).  A  suspension  of  plague  bacilli  is  made  by 
breaking  up  a  young  agar  culture  in  .75  per  cent  sodium  chloride 
solution;  the  larger  flocculi  of  growth  are  allowed  to  settle,  and 
the  fine,  supernatant  emulsion  is  employed  in  the  usual  way. 
According  to  the  results  of  the  German  Plague  Commission,  and 
the  observations  of  Cairns  made  during  the  Glasgow  epidemic,, 
it  may  be  said  that  the  reaction  is  best  obtained  with  dilutions  of 
the  serum  of  from  i  :  10  to  I  :  50.  Cairns  found  that  the  date  of 
its  appearance  is  about  a  week  after  the  onset  of  illness,  and  that 
it  usually  increases  till  about  the  end  of  the  sixth  week,  there- 
after fading  off.  It  is  most  marked  in  severe  cases,  character- 
ised by  an  early  and  favourable  crisis,  less  marked  in  severe 
cases  ultimately  proving  fatal,  whilst  in  very  mild  cases  it  is- 
feeble  or  may  be  absent.  The  method,  if  carefully  applied,  may 
be  of  service  under  certain  conditions ;  but  it  will  be  seen  that 
its  use  as  a  means  of  diagnosis  is  somewhat  restricted.  The 
serum  reaction  has  also  been  employed  to  distinguish  the  plague 
bacillus  from  other  organisms  morphologically  resembling  it. 

Methods  of  Diagnosis.  —  Where  a  bubo  is  present  a  little  of 
the  juice  may  be  obtained  by  plunging  a  sterile  hypodermic 


RELAPSING   FEVER.  447 

needle  into  the  swelling.  The  fluid  is  then  to  be  examined 
microscopically,  and  cultures  on  agar  or  blood  serum  should  be 
made  by  the  successive  stroke  method.  The  cultural  and  mor- 
phological characters  are  then  to  be  investigated,  the  most  impor- 
tant being  the  involution  forms  on  salt  agar  and  the  stalactite 
growth  in  bouillon,  though  the  latter  may  not  always  be  obtained 
with  the  plague  bacillus ;  the  pathogenic  properties  should  also 
be  studied,  the  guinea-pig  being  on  the  whole  most  suitable  for 
subcutaneous  inoculation.  In  many  cases  a  diagnosis  may  be 
made  by  microscopic  examination  alone,  as  in  no  other  known 
condition  than  plague  do  bacilli  with  the  morphological  char- 
acters of  the^  plague  bacillus  occur  in  the  lymphatic  glands.  An 
examination  of  the  blood  will  only  give  positive  results  in  severe 
cases.  And  in  every  instance,  on  the  occurrence  of  the  first 
suspected  case,  every  care  to  exclude  possibility  of  doubt  should 
be  used  before  a  positive  opinion  is'  given. 

In  a  case  of  suspected  plague  pneumonia,  in  addition  to 
microscopic  examination  of  the  sputum,  the  above  cultural 
methods  along  with  animal  inoculation  with  the  sputum  should 
be  carried  out;  subcutaneous  injection  in  the  guinea-pig  and 
smearing  the  nasal  mucous  membrane  of  the  rat  may  be  recom- 
mended. Here  a  positive  diagnosis  should  not  be  attempted  by 
microscopic  examination  alone,  especially  in  a  plague-free  dis- 
trict, as  bacilli  morphologically  resembling  the  plague  organism 
may  occur  in  the  sputum  in  other  conditions. 

RELAPSING  FEVER. 

At  a  comparatively  early  date,  namely  in  1873,  when  practi- 
cally nothing  was  known  with  regard  to  the  production  of  disease 
by  bacteria,  a  highly  characteristic  organism  was  discovered  in 
the  blood  of  patients  suffering  from  relapsing  fever.  This  discov- 
ery was  made  by  Obermeier,  and  the  organism  is  usually  known 
as  the  spirillum  or  spirochate  Obermeieri,  or  the  spirillum  of 
relapsing  fever.  He  described  its  microscopical  characters,  and 
found  that  its  presence  in  the  blood  had  a  definite  relation  to 
the  time  of  the  fever,  as  the  organism  rapidly  disappeared  about 
the  time  of  the  crisis,  and  reappeared  when  a  relapse  occurred. 
He  failed  to  find  such  an  organism  in  any  other  disease.  His 
observations  were  fully  confirmed,  and  his  views  as  to  its  causal 
relationship  to  the  disease  were  generally  accepted.  Later,  the 


448  RELAPSING   FEVER. 

disease  was  produced  in  the  human  subject  by  inoculations  with 
blood  containing  the  organisms,  and  a  similar  condition  has  been 
produced  in  apes. 

Characters  of  the  Spirillum.  —  The  organisms  as  seen  in  the 
blood  during  the  fever  are  delicate  spiral  filaments  which  have  a 
length  of  from  two  to  six  times  the  diameter  of  a  red  blood  cor- 
puscle. They  are,  however,  exceedingly  thin,  their  thickness 
being  much  less  than  that  of  the  cholera  spirillum.  They  show 
several  regular  sharp  curves  or  windings,  of  number  varying 
according  to  the  length  of  the  spirilla,  and  their  extremities  are 
finely  pointed  (Fig.  153).  They  are  actively  motile,  and  may 

be  seen  moving  quickly  across 
the  microscopic  field  with  a  pe- 
culiar movement  which  is  partly 

v  r  *~  ^JalnMili*  S* 

twisting  and  partly  undulatory, 
and  disturbing  the  blood  cor- 
y%ftgP^J\     puscles  in  their  course, 
jgftk  They     stain    with    watery 

solutions  of   the  basic  aniline 

^^^^^B§!  -i?^b 

dyes,  though  somewhat  faintly, 
anc^  are  best  coloured  in  film 
preparations    of    Lofrler's     or 
Kiihne's  methylene-blue    solu- 
FIG.  i53.-sPiriiia  of  relapsing  fever  in    tions.     When  thus  stained  they 
human  blood.    Film  preparation.  usually  have  a  uniform  appear- 

(After  Koch.)     x  about  1000. 

ance  throughout,  or  may  be 

slightly  granular  at  places,  but  they  show  no  division  into  short 
segments.  They  lose  the  stain  in  Gram's  method. 

In  blood  outside  the  body  the  organisms  have  a  considerable 
degree  of  vitality,  and  when  kept  in  sealed  tubes  have  been 
found  alive  and  active  after  many  days.  They  are  readily  killed 
at  a  temperature  of  60°  C,  but  may  be  exposed  to  o°  C.  without 
being  killed.  There  is  no  evidence  that  they  form  spores. 

Relations  to  the  Disease.  —  In  relapsing  fever,  after  a  period 
of  incubation,  there  occurs  a  rapid  rise  of  temperature  which  lasts 
for  about  five  to  seven  days.  At  the  end  of  this  time  a  crisis 
occurs,  the  temperature  falling  quickly  to  normal.  In  the  course 
of  about  other  seven  days  a  sharp  rise  of  temperature  again 
takes  place,  but  on  this  occasion  the  fever  lasts  a  shorter  time, 
again  suddenly  disappearing.  A  second  or  even  third  relapse 


RELATIONS   TO   THE   DISEASE.  449 

may  occur  after  a  similar  interval.  The  spirilla  begin  to  appear 
in  the  blood  shortly  before  the  onset  of  the  pyrexia,  and  during 
the  rise  of  temperature  rapidly  increase  in  number.  They  are 
very  numerous  during  the  fever,  a  large  number  being  often 
present  in  every  field  of  the  microscope  when  the  blood  is 
examined  at  this  stage.  They  begin  to  disappear  shortly  before 
the  crisis ;  after  the  crisis  they  are  entirely  absent  from  the 
circulating  blood.  A  similar  relation  between  the  presence  of 
the  spirilla  in  the  blood  and  the  fever  is  found  in  the  case  of 
the  relapses,  whilst  between  these  they  are  entirely  absent 
Munch  in  1876  produced  the  disease  in  the  human  subject  by 
injecting  blood  containing  the  spirilla,  and  this  experiment  has 
been  several  times  repeated  with  the  same  result ;  after  a  period 
of  incubation  the  spirilla  begin  to  appear  in  the  circulating  blood, 
and  their  appearance  is  soon  followed  by  the  occurrence  of 
pyrexia. 

Numerous  attempts  to  cultivate  this  organism  outside  the  body 
have  all  been  attended  with  failure,  and  it  has  been  abundantly 
shown  that  it  does  not  grow  on  any  of  the  media  ordinarily  in 
use.  Koch  found  that  on  blood  serum  the  filaments  of  the  spi- 
rilla increased  somewhat  in  length,  and  formed  a  sort  of  felted 
mass,  but  that  no  multiplication  took  place.  Additional  proof, 
however,  that  the  organism  is  the  cause  of  the  disease  has  been 
afforded  by  experiments  on  monkeys,  and  facts  of  considerable 
interest  have  been  thus  established.  Carter,  in  1879,  was  the 
first  to  show  that  the  disease  could  be  readily  produced  in  these 
animals,  and  his  experiments  were  confirmed  by  Koch.  In  such 
experiments  the  blood  taken  from  patients  and  containing  the 
spirilla  was  injected  subcutaneously.  In  the  disease  thus  pro- 
duced there  is  an  incubation  period  which  usually  lasts  about  three 
days.  At  the  end  of  that  time  the  spirilla  rapidly  appear  in  the 
blood,  and  shortly  afterwards  the  temperature  quickly  rises.  The 
period  of  pyrexia  usually  lasts  for  two  or  three  days,  and  is  fol- 
lowed by  a  marked  crisis.  As  a  rule  there  is  no  relapse,  but 
occasionally  one  of  short  duration  occurs.  The  presence  of  spi- 
rilla in  the  blood  has  the  same  relation  to  the  pyrexial  period  as 
in  the  human  subject. 

For  a  long  time  the  place  and  mode  of  destruction  of  the 
spirilla  were  quite  unknown;  but  valuable  light  was  thrown  on 
these  points  by  Metchnikoff,  who  produced  the  disease  in  mon- 

2  G 


450  RELAPSING   FEVER. 

keys  and  killed  them  at  various  stages  of  the  fever.  He  found 
that  during  the  fever  the  spirilla  were  practically  never  taken  up 
by  the  leucocytes  in  the  circulating  blood,  but  that  at  the  time  of 
the  crisis  on  disappearing  from  the  blood,  they  accumulated  in 
the  spleen  and  were  ingested  in  large  numbers  by  the  microphages 
or  polymorpho-nuclear  leucocytes.  Within  these  they  rapidly 
underwent  degeneration,  and  disappeared.  Metchnikoff  also 
found  that  after  the  spirilla  had  disappeared  from  the  blood, 
the  disease  could  be  produced  in  another  animal  by  inoculations 
with  spleen  pulp,  in  which  the  spirilla  were  contained  within  the 
leucocytes,  thus  showing  that  they  were  living  and  active  in  the 
spleen.  It  is  to  be  noted  in  this  connection  that  swelling  of 
the  spleen  is  a  very  marked  feature  in  relapsing  fever.  These 
observations  have  been  entirely  confirmed  by  Soudakewitch,  who 
also  showed  that  the  destruction  of  the  spirilla  in  the  spleen  of  a 
monkey  (Cercocebus  fuliginosus)  was  an  extremely  rapid  one,  as 
they  were  all  destroyed  ten  hours  after  their  disappearance  from 
the  blood.  He  also  produced  the  disease  in  two  monkeys  from 
which  the  spleen  had  been  previously  removed,  the  animals  hav- 
ing been  allowed  to  recover  completely  from  the  operation.  In 
these  cases  the  spirilla  did  not  disappear  from  the  blood  at  the 
usual  time,  but  rather  increased  in  number,  and  a  fatal  result 
followed  on  the  eighth  and  ninth  days  respectively.  Post  mortem 
he  found  the  spirilla  in  enormous  numbers  throughout  the  blood- 
vessels, and  in  the  portal  vein  they  almost  equalled  the  red  blood 
corpuscles  in  number.  These  experiments  would  appear  to  es- 
tablish the  important  function  of  the  spleen  in  the  destruction  of 
the  organisms ;  they  do  not  show,  however,  why  the  organisms 
disappear  from  the  blood  at  a  particular  time  and  accumulate  in 
the  spleen. 

Views  of  a  different  character  have  been  advanced  by  Lamb. 
According  to  this  observer,  while  in  the  monkey  (Macacus  radia- 
tus)  a  relapse  rarely  occurs,  this  animal  about  a  month  after 
recovery  is  susceptible  to  fresh  inoculation.  During  the  two  or 
three  weeks  following  an  attack  of  the  fever  it,  however,  mani- 
fests a  degree  of  immunity  to  infection.  If  in  such  an  animal 
the  spleen  be  excised,  it  still  does  not  suffer  from  the  disease 
after  fresh  inoculation.  The  immunity  Lamb  attributes  to  the 
presence  of  bactericidal  bodies  in  the  serum.  The  proof  of  this 
advanced  is  that  in  vitro  the  serum  brings  the  movements  of  the 


MALTA   FEVER. 

spirilla  to  an  end,  clumps  them,  and  causes  their  disintegration ; 
and  further,  that  when  the  spirilla  and  the  immune  serum  were 
injected  in  one  case  into  a  fresh  monkey  no  disease  developed. 
In  opposition  to  Soudakewitch,  Lamb  found  that  with  a  monkey 
from  which  the  spleen  had  been  removed  and  inoculation  prac- 
tised death  did  not  occur.  Here  it  is  to  be  noted,  however,  that 
the  animals  used  by  Soudakewitch  and  by  Lamb  were  of  differ- 
ent species. 

In  the  case  of  the  human  subject  it  has  been  found  that  a 
second  attack  of  the  disease  can  follow  the  first  after  a  com- 
paratively short  period  of  time,  and  it  is  often  said  that  one 
attack  does  not  confer  immunity.  It  is  probably  rather  the  case 
that  the  immunity  conferred  is  of  very  short  duration.  The 
course  of  events  in  the  disease  might  be  explained  by  supposing 
that  immunity  of  short  duration  is  produced  during  the  first 
period  of  pyrexia,  but  that  it  does  not  last  until  all  the  spirilla 
have  been  destroyed,  some  still  surviving  in  internal  organs. 
With  the  disappearance  of  the  immunity  the  organisms  reappear 
in  the  blood,  the  relapse  being,  however,  of  shorter  duration  and 
less  severe  than  the  first  attack.  This  is  repeated  till  the 
immunity  lasts  long  enough  to  allow  all  the  organisms  to  be 
killed.  Lamb's  observations  suggest  the  probability  that  this 
immunity  after  the  crisis  may  be  evidenced  by  bactericidal 
powers  in  the  serum,  and  according  to  the  recent  work  of 
Sawtschenko  and  Melkich,  there  are  developed  during  the  dis- 
ease both  an  immune  body  and  an  agglutinin  (vide  chapter  on 
Immunity).  The  former,  in  association  with  the  alexine  of  the 
blood  serum,  brings  about  a  bactericidal  effect,  whilst  by  itself  it 
also  constitutes  the  means  whereby  a  positive  chemiotaxis  is 
exerted  on  the  leucocytes.  It  is  further  to  be  noted  that  re- 
lapsing fever  is  unique  amongst  bacterial  diseases  affecting  the 
human  subject,  in  respect  of  the  enormous  numbers  of  organ- 
isms which  can  be  observed  in  the  circulating  blood  during  life. 

MALTA  FEVER. 

SYNONYMS:  —  MEDITERRANEAN  FEVER.     ROCK  FEVER  OF  GIBRALTAR. 
NEAPOLITAN  FEVER,  ETC. 

This  disease  is  of  common  occurrence  along  the  shores  of  the 
Mediterranean  and  in  its  islands.     By  means  of  the  agglutinating 


452  MALTA   FEVER. 

test  Wright  and  Smith  have  shown  that  it  occurs  also  in  some 
parts  of  India,  and  it  has  also  been  observed  in  the  United  States 
and  Porto  Rico  (W.  I.).  There  can  be  little  doubt  that  its  dis- 
tribution will  be  found  to  be  much  wider  than  was  formerly  sup- 
posed. Although  from  its  symptomatology  and  pathological 
anatomy  it  had  been  recognised  as  a  distinct  affection,  and  was 
known  under  various  names,  its  precise  etiology  was  unknown 
till  the  publication  of  the  researches  of  Surgeon-Major  Bruce 
in  1887.  From  the  spleen  of  patients  dead  of  the  disease  he 
cultivated  a  characteristic  organism,  now  known  as  the  micrococ- 
cus  melitensis  >  and  by  means  of  inoculation  experiments  estab- 
lished its  causal  relationship  to  the  disease.  His  results  have 
been  confirmed  by  other  observers,  and  additional  confirmatory 
evidence  has  been  supplied  by  means  of  serum  diagnosis,  as 
will  be  described  below.  Bacteriological  methods  have  there- 
fore been  the  means  of  differentiating  the ,  disease,  and  also  of 
affording  a  more  exact  basis  for  diagnosis. 

The  duration  of  the  disease  is  usually  long  —  often  two  or 
three  months,  though  shorter  and  much  longer  periods  are  met 
with.  Its  course  is  very  variable,  the  fever  being  of  the  con- 
tinued type  with  irregular  remissions.  In  addition  to  the  usual 
symptoms  of  pyrexia  there  occur  profuse  perspirations,  pains,  and 
sometimes  swellings  in  the  joints,  occasionally  orchitis,  whilst 
constipation  is  usually  a  marked  feature.  The  mortality  is  low 
—  about  2  per  cent  (Bruce). 

In  fatal  cases  the  most  striking  post-mortem  change  is  in  the 
spleen.  This  organ  is  enlarged,  often  weighing  slightly  over  a 
pound,  and  in  a  condition  of  acute  congestion ;  the  pulp  is  soft 
and  may  be  diffluent,  and  the  Malpighian  bodies  are  swollen  and 
indistinct.  In  the  other  organs  the  chief  change  is  cloudy  swell- 
ing ;  in  the  kidneys  there  may  be  in  addition  glomerular  nephri- 
tis. The  lymphoid  tissue  of  the  intestines  shows  none  of  the 
changes  characteristic  of  typhoid  fever. 

Micrococcus  melitensis.  —  This  is  a  small,  rounded  or  slightly 
oval  organism  about  .5  /u,  in  diameter,  which  is  specially  abundant 
in  the  spleen.  It  usually  occurs  singly  or  in  pairs,  but  in  cultures 
short  chains  are  also  met  with  (Fig.  154).  (Durham  has  shown 
that  in  old  cultures  kept  at  the  room  temperature  bacillary  forms 
appear,  and  we  have  noticed  indications  of  such  in  comparatively 
young  cultures;  the  usual  form  is,  however,  that  of  a  coccus.) 


CULTIVATION   OF   MIC.  MELITENSIS.  453 

It  stains  fairly  readily  with  the  ordinary  basic  aniline  stains,  but 
loses  the  stain  in  Gram's  method.     It  is  generally  said  to  be 
a   non-motile    organism.     Gor- 
don, however,  is  of  a  contrary  ,  v  - 
opinion,  and  has  recently  dem- 
onstrated that  it  possesses  from                      **     •  ^ 
one  to  four  flagella,  which,  how-        ,  \  •"  .    '  *%     *\  ,  •    '     '*       *• 
ever,  are  difficult  to  stain.     In     .  •*  \%    /   %  ¥%  *     ..  * 
the  spleen  of   a  patient  dead                *     t*j*  *     ''      ^ 
of  the  disease  it  occurs  irregu-          f*      ^  ' '  *     ".x  .. 
larly     scattered     through     the        -.             .*•*      % 
congested  pulp.     It  may  also                          *,  * .    , 

be    found    in    small    numbers  "V        *,       » :- 

".       • 

post  mortem  in  the  capillaries 

Of    Various    Organs,    but    exami-  FIG.  iS4.  —  Micrococcus  melitensis,  from 

nation     of      the      blood      during     a  two  days' culture  on  agar  at  37°  C. 
.  .  Stained  with  fuchsin.     x  1000. 

life  gives  negative  results.     It 

can,   however,  be  obtained  by  puncture  of  the  spleen  during 

life. 

Cultivation.  —  This  can  usually  readily  be  effected  by  making 
stroke-cultures  on  agar  tubes  from  the  spleen  pulp  and  incu- 
bating at  37°  C.  The  colonies,  which  are  usually  not  visible 
before  the  third  day,  appear  as  small  round  discs,  slightly  raised 
and  of  somewhat  transparent  appearance.  The  maximum  size 
—  2-3  mm.  in  diameter  —  is  reached  about  the  ninth  day;  at 
this  period  by  reflected  light  they  appear  pearly  white,  while  by 
transmitted  light  they  have  a  yellowish  tint  in  the  centre,  bluish 
white  at  the  periphery.  A  stroke-culture  shows  a  layer  of 
growth  of  similar  appearance  with  somewhat  serrated  margins. 
Old  cultures  assume  a  buff  tint.  The  optimum  temperature  is 
37°  C.,  but  growth  still  occurs  down  to  about  20°  C.  On  gelatin 
at  summer  temperature  growth  is  extremely  slow  —  after  two  or 
three  weeks,  in  a  puncture  culture,  there  is  a  delicate  line  of 
growth  along  the  needle  track  and  a  small  flat  expansion  of 
growth  on  the  surface.  There  is  no  liquefaction  of  the  medium. 

In  bouillon  there  occurs  a  general  turbidity  with  flocculent 
deposit  at  the  bottom ;  on  the  surface  there  is  no  formation  of 
a  pellicle.  On  potatoes  no  visible  growth  takes  place  even  at 
the  body  temperature,  though  the  organism  multiplies  to  a  certain 
extent. 


454  MALTA   FEVER. 

Relations  to  the  Disease.  —  There  is  in  the  first  place  ample 
evidence,  from  examination  of  the  spleen,  both  post  mortem  and 
during  life,  that  this  organism  is  always  present  in  the  disease. 
The  experiments  of  Bruce  and  Hughes  show  that  by  inoculation 
with  even  comparatively  small  doses  of  pure  cultures  the  dis- 
ease can  be  produced  in  monkeys.  In  these  experiments  seven 
animals  in  all  were  used,  in  every  case  with  a  positive  result. 
Four  died  at  varying  periods  of  time,  after  showing  well-marked 
fever,  closely  resembling  in  character  that  occurring  in  the 
human  subject,  and  the  micrococcus  was  obtained  from  the 
organs  post  mortem.  The  other  three  animals  recovered  after 
suffering  from  illness  with  corresponding  pyrexia  —  in  two  cases 
extending  over  two  months.  The  disease  has  also  been  pro- 
duced in  the  human  subject  by  accidental  inoculation  with  a 
pure  culture  of  the  micrococcus,  Birt  and  Lamb  citing  three 
cases,  and  Strong  and  Musgrave  one  case. 

Rabbits,  guinea-pigs,  and  mice  are  insusceptible  to  inocula- 
tion by  the  ordinary  method.  .  Durham,  by  using  the  intra- 
cerebral  method  of  inoculation,  has,  however,  succeeded  in 
raising  the  virulence  so  that  the  organism  is  capable  of  produc- 
ing in  guinea-pigs  on  intraperitoneal  injection  illness  with  some- 
times a  fatal  result  many  weeks  afterwards.  An  interesting 
point  brought  out  by  these  experiments  is  that  in  the  case  of 
animals  which  survive  the  micrococcus  may  be  cultivated  from 
the  urine  several  months  after  inoculation. 

Agglutinative  Action  of  Serum.  —  The  blood  serum  of  patients 
suffering  from  Malta  fever  possesses  the  power  of  agglutinating 
the  micrococcus  melitensis  in  a  manner  analogous  to  what  has 
been  described  in  the  case  of  typhoid  fever.  This  action  is 
manifested  throughout  the  disease,  and  also  for  a  considerable 
time  after  recovery.  Wright  and  Smith  found  it  well  marked  a 
year  afterwards.  They  found  that  the  greatest  dilution  which 
gives  distinct  agglutinative  effects  varies  in  different  cases  from 
I  :  10  to  i  :  1000.  As  regards  relation  to  prognosis,  the  observa- 
tions of  Birt  and  Lamb  and  of  Bassett-Smith  have  given  results 
analogous  to  those  obtained  in  typhoid  (p.  343). 

Methods  of  Diagnosis.  —  During  life  the  best  means  of  diag- 
nosis is  supplied  by  the  agglutinative  test  just  described  (for 
technique,  vide  p.  109). 

Cultures  are  most  easily  obtained   from   the    spleen    either 


YELLOW   FEVER.  455 

during  life  or  post  mortem.  Inoculate  a  number  of  agar  tubes 
by  successive  strokes  and  incubate  at  37°  C.  Film  preparations 
should  also  be  made  from  the  spleen  pulp  and  stained  with 
carbol-thionin-blue  or  diluted  carbol-fuchsin  (i  :  10). 

YELLOW  FEVER. 

Yellow  fever  is  an  infectious  disease  which  is  endemic  in  the 
West  Indies,  in  Brazil,  in  Sierra  Leone,  and  the  adjacent  parts 
of  West  Africa,  though  it  is  probable  that  it  was  from  the  first- 
named  region  that  the  others  were  originally  infected.  From 
time  to  time  serious  outbreaks  occur,  during  which  neighbour- 
ing countries  also  suffer,  and  the  disease  may  be  carried  to  other 
parts  of  the  world.  In  this  way  epidemics  have  occurred  in  the 
United  States,  in  Spain,  and  even  in  England,  infection  usually 
being  carried  by  cases  occurring  among  the  crews  of  ships.  In 
the  parts  where  it  is  endemic,  though  usually  a  few  cases  may 
occur  from  time  to  time,  there  is  some  evidence  that  occasion- 
ally the  disease  may  remain  in  abeyance  for  many  years  and 
then  originate  de  nova.  There  is,  therefore,  reason  to  suspect 
that  the  infective  agent  can  exist  for  considerable  periods  out- 
side the  human  body.  It  is  possible,  however,  that  continuity 
may  be  maintained  by  the  occurrence  of  a  mild  type  of  the 
disease  which  may  be  grouped  with  the  "  bilious  fevers  "  preva- 
lent in  yellow-fever  regions.  This  would  explain  the  degree  of 
immunity  which  is  shown  during  a  serious  epidemic  by  the 
older  immigrants. 

Great  variations  are  observed  in  the  clinical  types  under 
which  the  disease  presents  itself.  Usually  after  from  two  to  six 
days'  incubation  a  sudden  onset  in  the  form  of  a  rigour  occurs. 
The  temperature  rises  to  104°- 105°  F.  The  person  is  livid,  with 
outstanding  bloodshot  eyes.  There  are  present  great  prostra- 
tion, pain  in  the  back,  and  vomiting,  at  first  of  mucus,  later  of 
bile.  The  urine  is  diminished  and  contains  albumin.  About 
the  fifth  day  an  apparent  improvement  takes  place,  and  this 
may  lead  on  to  recovery.  Frequently,  however,  the  remission, 
which  may  last  from  a  few  hours  to  two  days,  is  followed  by 
an  aggravation  of  all  the  symptoms.  The  temperature  rises, 
jaundice  is  observed,  haemorrhages  occur  from  all  the  mucous 
surfaces,  causing,  in  the  case  of  the  stomach,  the  "black  vomit" 


456  YELLOW   FEVER. 

—  one  of  the  clinical  signs  of  the  disease  in  its  worst  form. 
Anuria,  coma,  and  cardiac  collapse  usher  in  a  fatal  issue.  The 
mortality  varies  in  different  epidemics  from  about  35  to  99  per 
cent  of  those  attacked.  Both  white  and  black  races  are  sus- 
ceptible, but  those  who  have  resided  long  in  a  country  are  less 
susceptible  than  new  immigrants.  An  attack  of  the  disease 
usually  confers  complete  immunity  against  subsequent  infection. 
Post  mortem  the  stomach  is  found  in  a  state  of  acute  gastritis, 
and  contains  much  altered  blood  derived  from  haemorrhages 
which  have  occurred  in  the  mucous  and  submucous  coats.  The 
intestine  may  be  normal,  but  is  often  congested  and  may  be 
ulcerated  ;  the  mesenteric  glands  are  enlarged.  The  liver  is  in 
a  state  of  fatty  degeneration  of  greater  or  less  degree,  but  often 
resembling  the  condition  found  in  phosphorus  poisoning.  The 
kidneys  are  in  a  state  of  intense  glomerulo-nephritis,  with  fatty 
degeneration  of  the  epithelium.  There  is  congestion  of  the 
meninges,  especially  in  the  lumbar  region,  and  haemorrhages 
may  occur.  The  other  organs  do  not  show  much  change, 
though  small  haemorrhages  under  the  skin  and  into  all  the  tissues 

of  the  body  are  not  infrequent. 
In  the  blood  a  feature  is  the 

ff,  m  ^^  *       »-  excess  of  urea  present,  amount- 

ing,   it  may  be,    to  nearly  4 
/     ^,  per  cent. 

'    •  J&  *      "J^  The    Etiology    of    Yellow 

"*  ^t.4"  ***&        -£/    *         Fever.  —  Two  chief  views  are 
••  ^/;:/  here  held,     (i)  That  the  dis- 

+    '     '      ease  is  caused  by  a  bacterium 


~:"  '  '-,•  '  belonging  to  the  B.  coli  group 

\  t          ,  t^  and  called  the   B.   icteroides. 

(2)  That  the  causal  agent  is 
of    such   small  size    as  to  be 

FlG.    155.  —  Bacillus    icteroides,    from    a          .  •,-,        •        •    ••,-,  •> 

young  culture  on  agar  (Sanarelli).     x  1000.        microscopically    invisible,    and 

that  for  the  transmission  of  the 

disease  from  man  to  man  a  mosquito  acting  as  an  intermediate 
host  is  necessary.  This  latter  view,  which  chronologically  is 
second,  now  holds  the  more  important  position. 

i.  Bacillus  icteroides.  —  A  very  full  research  into  the  bacteriology  of  yellow 
fever  was  that  of  Sternberg  (1890),  the  result  of  which  was  that  of  various 
organisms  isolated  one  which  he  named  "  bacillus  x  "  appeared  possibly  to 


BACILLUS    ICTEROIDES. 


457 


have  some  causal  relationship  to  the  disease.     Sanarelli,  in  1897,  obtained 
cultures  of  an  organism  which  he  named  bacillus  icteroides,  and  which  he 
considers  to  be  the  cause  of  yellow  fever.     It  is  not  identical 
with  the  "bacillus  x,"  the  latter  being  a  variety  of  B.  coli. 

This  organism  has  rounded  ends,  is  2-4  /A  long,  about 
.5  fj.  broad,  often  occurring  in  pairs  (Fig.  155),  staining  by 
the  ordinary  stains  but  decolorising  by  Gram's  method.  It 
is  motile  and  possesses  four  to  eight  flagella.  It  grows  on  all 
the  usual  media ;  on  gelatin  plates  after  twenty-four  hours  the 
colonies  are  minute  points  somewhat  transparent  to  the  naked 
eye,  and  under  a  low  power  have  a  finely  granular  appearance. 
After  six  or  seven  days  there  appears  somewhere  in  the  colony 
a  focus  of  more  active  growth,  forming  an  opaque  centre,  from 
which  granular  striae  radiate  to  the  periphery.  The  gelatin  is 
not  liquefied.  The  most  characteristic  growth  is  that  on  sloped 
agar.  After  twenty-fours  at  37°  C.  there  is  a  grey  iridescent 
and  somewhat  transparent  growth.  On  being  transferred  to 
a  temperature  of  20°  C.  to  28°  C.  this  becomes  in  twelve 
hours  surrounded  by  a  halo  of  white,  opaque,  pearly  growth 
of  higher  .level  than  the  central  part.  In  a  few  days  the 
growth  at  the  lower  temperature  becomes  liquid  in  character 
and  runs  slowly  down  the  medium  as  a  drop  of  melted 
paraffin  would  do  (Fig.  156).  Growth  also  takes  place  in 
bouillon  and  blood  serum.  On  potatoes  there  is  a  fine  trans- 
parent pellicle  which  does  not  alter  its  colour  with  age.  Lit- 
mus milk  is  rendered  faintly  acid,  the  cream-ring  turning 
gradually  blue,  and  after  a  lapse  of  several  days  the  acid 
reaction  of  the  medium  gives  place  slowly  to  an  alkaline 
change;  no  coagulation  occurs.  The  bacillus  ferments  glu- 
cose, but  not  lactose  or  saccharose.  It  occasionally  gives  a 
feeble  indol  reaction. 

Sanarelli  investigated  twelve  cases  of  yellow  fever  and 
found  the  B.  icteroides  present  in  relatively  small  numbers 
in  six.  It  appeared  chiefly  in  the  capillaries  of  the  liver  and 
kidneys,  rarely  in  other  parts  of  the  body.  It  was  never  found 
in  the  gastro-intestinal  tract. 

Inoculation    experiments  did  not  give   characteristic   re- 
sults, but  Sanarelli  states  that  sterile  bouillon  cultures,  when 
injected  subcutaneously  or  intravenously  in  man,  give  rise  to  all  the  symptoms 
of  yellow  fever. 

According  to  Sanarelli7s  view  the  bacillus  is  to  be  looked  on  as  settling 
chiefly  in  the  liver  and  kidneys  and  there  producing  very  powerful  toxins 
whose  chief  effects  are  on  the  cells  of  these  organs  and  on  the  small  blood- 
vessels of  the  body,  thus  causing  the  rupture  of  vessel  walls  which  frequently 
results,  and  opening  up  a  path  for  infection  by  other  organisms  which  may 
produce  secondary  infections.  Sanarelli  states  that  the  serum  of  yellow-fever 
patients  clumps  the  B.  icteroides  in  a  dilution  of  i :  40.  The  reaction  is  said 
to  appear  on  the  second  day. 


FIG.  156.  — 
Culture  of  Ba- 
cillus icteroides 
on  agar,  show- 
ing the  charac- 
teristic appear- 
ance  when 
incubated  at 
the  two  tem- 
peratures men- 
tioned (Sana- 
relli). Natural 
size. 


458  YELLOW   FEVER. 

The  results  of  Sanarelli  were  certainly  striking,  but  they  have  not  been 
confirmed  by  recent  observations.  Of  the  investigators  immediately  following 
Sanarelli,  some  stated  that  they  found  the  B.  icteroides  in  a  certain  proportion 
of  cases,  whilst  others  obtained  negative  results. 

Reed  and  Carroll  regard  this  organism  as  identical  with  the  bacillus  of 
hog  cholera;  and  culturally,  it  parallels  the  growth  characteristics  of  B. 
enteritidis  (Gaertner). 

2.  The  Mosquito  Theory.  —  The  most  extensive  and  carefully 
planned  inquiry  into  the  etiology  of  yellow  fever  has  been  that 
of  the  United  States  Army  Commission  (1900-1),  and  the  result 
of  their  labours  has  been  the  bringing  forward  of  an  entirely 
new  order  of  facts.  In  the  first  place,  they  failed  to  find  the  B. 
icteroides  in  the  blood  of  patients  suffering  from  the  disease ;  in 
twenty-four  cases  blood  was  withdrawn  from  a  vein  and  cultures 
made  on  various  media  with  negative  result.  Yet  they  found 
that  a  small  quantity  of  such  blood  (.5-2  c.c.),  when  injected  into 
a  healthy  subject,  was  sufficient  to  produce  the  disease.  Further- 
more, in  three  instances  they  found  that  the  blood  serum  of  a 
yellow-fever  patient,  diluted  and  passed  through  a  Berkefeld 
filter,  still  retained  its  pathogenic  properties,  but  it  was  found 
that  if  the  pure  or  diluted  serum,  either  filtered  or  unfiltered, 
were  heated  for  ten  minutes  at  55°C.  and  injected  in  quantities 
of  1.5  c.c.,  it  was  perfectly  innocuous. 

From  this  result,  and  from  the  negative  result  of  microscopic 
examination,  they  surmised  that  the  virus  was  not  one  of  the 
ordinary  bacteria  and  was  probably  ultra-microscopic  in  nature.1 
The  next  important  conclusion  arrived  at  was  that  the  disease 
is  not  communicable  by  direct  contact  with  those  suffering  from 
the  disease,  with  their  fomites,  etc.  In  a  specially  constructed 
house  seven  men  were  exposed  to  the  most  intimate  contact 
with  the  fomites  of  yellow-fever  patients  for  a  period  of  twenty 
days  each,  the  soiled  garments  worn  by  the  patients  being  in 
some  instances  actually  slept  in  by  these  men;  the  result  was 
that  not  one  of  those  thus  exposed  contracted  the  disease.  By 
far  the  most  important  result  of  this  investigation,  however,  is 
the  establishment  of  the  part  played  by  mosquitoes  in  the  trans- 
mission of  the  disease.  Of  twelve  non-immune  persons  who 

1  In  several  diseases  the  existence  of  such  causal  factors  is  suspected.  The  other 
examples  are  foot-and-mouth  disease,  South  African  horse-sickness,  and  the  contagious 
pleuro-pneumonia  of  cattle. 


THE   MOSQUITO    THEORY.  459 

voluntarily  allowed  themselves  to  be  bitten  with  mosquitoes 
which  had  previously  fed  on  the  blood  of  yellow-fever  patients, 
ten  took  the  disease,  the  period  of  incubation  being  from  three 
to  six  days.  Two  of  the  men  who  were  thus  infected  had  been 
previously  exposed  to  contact  with  fomites  without  result.  The 
species  of  mosquito  found  capable  of  carrying  the  infection  in 
this  way  is  the  Stegomyia  fasciata.  It  was  found  that  a  period 
of  about  twelve  days  must  elapse  after  the  insect  bites  a  patient 
suffering  from  yellow  fever  before  it  becomes  infective  to  another 
subject,  and,  on  the  other  hand,  that  it  retains  the  power  of  infec- 
tion for  nearly  sixty  days  later.  These  results  have  been  con- 
firmed by  Guiteras,  whose  investigation  was  carried  out  along 
similar  lines ;  of  seventeen  individuals  bitten  by  the  infected 
stegomyia,  eight  took  yellow  fever  and  of  these  three  died. 

As  yet  nothing  has  been  determined  by  these  workers  regard- 
ing the  nature  of  the  virus,  but  the  results  already  obtained  have 
supplied  the  basis  for  preventive  measures  against  the  disease, 
these  being  directed  towards  the  destruction  of  mosquitoes  and 
the  protection  of  those  suffering  from  yellow  fever,  and  also  the 
healthy,  against  the  bites  of  these  insects.  Already  a  striking 
degree  of  success  has  been  obtained  in  Havana..  Such  measures 
came  into  force  in  February,  1901,  and  in  ninety  days  the  town 
was  free  of  yellow  fever  and  for  fifty-four  days  later  no  new 
cases  occurred.  And  although  subsequently  the  disease  was 
reintroduced  into  the  town,  no  difficulty  was  experienced  in 
stamping  it  out  by  the  same  measures.  It  would  be  unsafe,  as 
yet,  to  generalise  from  this  particular  instance,  but  there  is 
a  good  prospect  that  at  least  a  measure  of  similar  success  will 
be  attained  in  other  places. 


CHAPTER   XX. 

IMMUNITY. 

Introductory.  —  By  immunity  is  meant  non-susceptibility  to  a 
given  disease  or  to  a  given  organism,  either  under  natural  con- 
ditions or  under  conditions  experimentally  produced.  The  term 
is  also  used  in  relation  to  the  toxins  of  an  organism.  Immunity 
may  be  possessed  by  an  animal  naturally,  and  is  then  usually 
called  natural  immunity,  or  it  may  be  acquired  by  an  animal 
either  by  passing  through  an  attack  of  the  disease  or  by  artificial 
means  of  inoculation.  It  has  been  shown  that  certain  diseases 
affect  the  lower  animals  but  never  occur  in  the  human  subject, 
e.g.  swine  plague  ;  and,  on  the  other  hand,  diseases  such  as 
typhoid  fever  and  cholera  do  not  under  natural  conditions  affect 
any  of  the  lower  animals,  so  far  as  is  known.  That  is  to  say, 
man  and  the  lower  animals  respectively  enjoy  immunity  against 
certain  diseases,  when  exposed  to  infection  under  ordinary  con- 
ditions. From  this  fact,  however,  it  does  not  follow  that  when 
the  organisms  of  the  respective  diseases  are  introduced  into  the 
body  by  artificial  methods  of  inoculation,  pathological  effects 
will  not  follow.  We  have  seen  above,  for  example,  that  the 
organisms  of  cholera  and  typhoid  may  artificially  be  made  to  in- 
fect guinea-pigs,  though  they  do  not  do  so  naturally.  Immunity 
may  thus  be  of  very  varying  degrees,  and  accordingly  the  use 
of  the  term  has  a  correspondingly  relative  significance.  Such  a 
thing  as  absolute  immunity  is  scarcely  known,  just  as  we  have 
seen  in  the  case  with  absolute  susceptibility.  This  is  not  only 
true  of  infection  by  bacteria,  but  of  toxins  also  ;  —  when  the  re- 
sistance of  an  animal  to  these  is  of  high  degree,  the  resistance 
may  be  overcome  by  a  very  large  dose  of  the  toxic  agent.  For 
example,  the  common  fowl  may  be  able  to  resist  as  much  as 
20  c.c.  of  powerful  tetanus  toxin,  but  on  this  amount  being  ex- 
ceeded may  be  affected  by  tetanic  spasms  (Klemperer).  On 
the  other  hand,  in  cases  where  the  natural  powers  of  resistance 

460 


ACQUIRED    IMMUNITY.  461 

are  very  high,  these  can  be  still  further  exalted  by  artifi- 
cial means,  that  is,  the  natural  immunity  may  be  artificially 
intensified. 

Acquired  Immunity  in  the  Human  Subject.  —  The  following 
facts  are  supplied  by  a  study  of  the  natural  diseases  which  affect 
the  human  subject.  First,  in  the  case  of  certain  diseases,  one 
attack  protects  against  another  for  many  years,  sometimes 
practically  for  a  lifetime,  e.g.  smallpox,  typhoid,  scarlet  fever, 
etc.  Secondly,  in  the  case  of  other  diseases,  e.g.  erysipelas, 
diphtheria,  influenza,  and  pneumonia,  a  patient  may  suffer  from 
several  attacks.  In  the  case  of  the  diseases  of  the  second  group, 
however,  experimental  research  has  shown  that  in  many  of  them 
a  certain  degree  of  immunity  does  follow ;  and,  though  we  can- 
not definitely  state  it  as  a  universal  law,  it  must  be  considered 
highly  probable  that  the  passing  through  an  attack  of  an  acute 
disease  produced  by  an  organism,  confers  immunity  for  a  longer 
or  shorter  period.  The  immunity  is  not,  however,  to  be  regarded 
as  the  result  of  the  disease  per  se,  but  of  the  bacterial  products 
introduced  into  the  system ;  as  will  be  shown  below,  by  suitable 
gradation  of  the  doses  of  such  products,  or  by  the  use  of  weakened 
toxins,  a  high  degree  of  immunity  may  be  attained  without  the 
occurrence  of  any  symptoms  whatever. 

The  facts  known  regarding  vaccination  and  smallpox  ex- 
emplify another  principle.  We  may  take  it  as  practically  proved 
that  vaccinia  is  variola  or  smallpox  in  the  cow,  and  that  when 
vaccination  is  performed,  the  patient  is  inoculated  with  a  modified 
variola  (vide  Smallpox,  in  Appendix).  Vaccination  produces 
certain  pathogenic  effects  which  are  of  trifling  degree  as  com- 
pared with  those  of  smallpox,  and  we  find  that  the  degree  of 
protection  is  less  complete  and  lasts  a  shorter  time  than  that 
produced  by  the  natural  disease.  Again,  inoculation  with  lymph 
from  a  smallpox  pustule  produces  a  form  of  smallpox  less  severe 
than  the  natural  disease  but  a  much  more  severe  condition  than 
that  produced  by  vaccination,  and  it  is  found  that  the  degree  of 
protection  or  immunity  resulting  occupies  an  intermediate  posi- 
tion. The  corresponding  'general  conclusion  from  experiments 
is  that  the  more  virulent  the  organism  injected,  provided  that 
the  animal  recovers  satisfactorily,  the  higher  is  the  degree  of 
immunity  acquired  by  it  against  that  organism.  Thus  in  develop- 
ing immunity  of  the  highest  degree  the  most  virulent  organisms 


462  IMMUNITY. 

are  ultimately  employed.     A  corresponding  principle,  with  cer- 
tain restrictions  (vide  p.  4/1),  obtains  in  the  case  of  toxins. 

Immunity  and  Recovery  from  Disease.  —  Recovery  from  an 
acute  infective  disease  shows  that  in  natural  conditions  the  virus 
may  be  exhausted  after  a  time,  the  period  of  time  varying  in 
different  diseases.  How  this  is  accomplished  we  do  not  yet 
fully  know,  but  it  has  been  found  in  the  case  of  diphtheria, 
typhoid,  cholera,  pneumonia,  etc.,  that  in  the  course  of  the 
disease  certain  substances  (called  by  German  writers  Antikorper) 
appear  in  the  blood,  which  are  antagonistic  either  to  the  toxin 
or  to  the  vital  activity  of  the  organism.  In  such  cases  a  process 
of  immunisation  would  appear  to  be  going  on  during  the  prog- 
ress of  the  disease,  and  when  this  immunisation  has  reached  a 
certain  height,  the  disease  naturally  comes  to  an  end.  It  can- 
not, however,  be  said  as  yet  that  such  antagonistic  substances 
are  developed  in  all  cases  ;  and  it  is  by  no  means  the  case  that 
the  degree  of  immunity  (active)  is  always  proportional  to  the 
amount  of  these  substances  in  the  blood. 

ARTIFICIAL  IMMUNITY. 

Varieties.  —  A  number  of  facts  regarding  immunity  have 
been  given  in  the  description  of  the  pathogenic  organisms  in 
previous  chapters.  We  shall  here  give  a  general  systematic 
description  of  the  methods,  and  discuss  the  principles  involved. 
According  to  the  means  by  which  it  is  produced,  immunity  may 
be  said  to  be  of  two  kinds,  to  which  the  terms  active  "&&&  passive 
are  generally  applied,  or  we  may  speak  of  immunity  directly,  or 
indirectly  produced.  We  shall  first  give  an  account  of  the 
established  facts,  and  afterwards  discuss  some  of  the  theories 
which  have  been  brought  forward  in  explanation  of  these  facts. 

Active  immunity  is  obtained  by  (a)  injections  of  the  organisms 
either  in  an  attenuated  condition  or  in  sub-lethal  doses,  or  (b)  by 
sub-lethal  doses  of  their  products,  i.e.  of  their  "toxins,"  the 
word  being  used  in  the  widest  sense.  By  repeated  injections 
at  suitable  intervals  the  dose  of  organisms  or  of  the  products 
can  be  gradually  increased ;  or,  what  practically  amounts  to  the 
same,  an  organism  of  greater  virulence  or  a  toxin  of  greater 
strength  may  be  used.  A  proportionate  degree  of  resistance  or 
immunity  can  thus  be  developed,  which  degree  in  course  of  time 


ARTIFICIAL   IMMUNITY.  463 

may  reach  a  very  high  level.  Such  a  method  can  be  preventive, 
but  it  can  never  be  curative,  as  the  immunity  must  be  developed 
before  the  onset  of  the  disease.  Immunity  of  this  kind  is  com- 
paratively slowly  produced  and  lasts  a  considerable  time,  the 
duration  varying  in  different  cases. 

Passive  immunity  depends  upon  the  fact  that  if  an  animal 
be  immunised  to  a  very  high  degree  by  the  previous  method,  its 
serum  may  have  distinctly  antagonistic  or  neutralising  effects 
when  injected  into  another  animal  along  with  the  organisms,  or 
with  their  products,  as  the  case  may  be.  Here  the  serum  of  the 
highly  immunised  animal  may  confer  immunity  on  another  ani- 
mal, if  introduced  at  the  same  time  as  infection  occurs  or  even  a 
short  time  afterwards ;  the  method  can,  therefore,  be  employed 
as  a  curative  agent.  The  serum  is  also  preventive,  i.e.  protects 
an  animal  from  subsequent  infection,  but  the  immunity  thus  con- 
ferred lasts  a  comparatively  short  time.  These  facts  form  the 
basis  of  serum  therapeutics.  When  such  a  serum  has  the  power 
of  neutralising  a  toxin  it  is  called  antitoxic  ;  when,  with  little  or 
no  antitoxic  power,  it  protects  against  the  living  bacterium  in 
a  virulent  condition,  it  is  called  antimicrobic  or  antibacterial 
(vide  infra). 

In  the  accompanying  table  a  sketch  of  the  chief  methods  by 
which  an  immunity  may  be  artificially  produced  is  given.  It 
has  been  arranged  for  purposes  of  convenience  and  to  aid  subse- 
quent description,  and  it  is  not  to  be  inferred  that  all  the  differ- 
ent methods  imply  essentially  different  principles.  There  is 
still  some  doubt  as  regards  the  relation  of  A  2,  for  example,  to 
A  i  and  A  3.  It  will  presently  be  seen  that  in  the  production 
of  immunity  it  is  to  be  noted  that  the  method  to  be  chosen 
usually  depends  on  the  individual  organism  against  which  im- 
munity is  to  be  conferred.  Thus  the  injection  of  diphtheria 
bacilli  will  immunise  both  against  subsequent  infection  by  bacilli 
and  against  the  injection  of  diphtheria  toxin,  and  immunisation 
by  diphtheria  toxin  will  have  a  similar  effect. 

ARTIFICIAL  IMMUNITY. 

A.  Active  Immunity  —  i.e.  produced  in  an  animal  by  an  in- 
jection, or  by  a  series  of  injections,  of  non-lethal  doses  of 
an  organism  or  its  toxins. 


464  IMMUNITY. 

1.  By  injection  of  the  living  organisms. 

(a)  Attenuated  in  various  ways.     Examples  :  — 

(1)  By  growing  in  the  presence  of  oxygen,  or  in  a 

current  of  air. 

(2)  By  passing   through   the   tissues    of    one   species 

of    animal    (becomes    attenuated    for    another 
species). 

(3)  By  growing  at  abnormal  temperatures,  etc. 

(4)  By  growing  in  the  presence  of  weak  antiseptics,  or 

by  injecting  the  latter  along  with  the  organism, 
etc. 

(b)  In  a  virulent  condition,  in  non-lethal  doses. 

2.  By  injection  of  the  dead  organisms. 

3.  By  injection  of  filtered  bacterial  cultures,  i.e.  toxins  ;  or  of 

cJiemical  substances  derived  from  these. 
These  methods  may  also  be  combined  in  various  ways. 
B.    Passive  Immunity  —  i.e.  produced  in  one  animal  by  injection 
of  the  serum  of  another  animal  highly  immunised  by  the 
methods  of  A. 

1.  By  antitoxic  serum,  i.e.  the  serum  of  an  animal  highly 

immunised  against  a  particular  toxin. 

2.  By  antibacterial  serum,  i.e.  the  serum  of  an  animal  highly 

immunised  against  a  particular  bacterium  in  the  living 
and  virulent  condition. 

A.    Active  Immunity. 

i.  By  Living  Cultures. — (a)  Attenuated.  —  In  the  earlier 
work  on  immunity  in  the  case  of  anthrax,  chicken  cholera,  swine 
plague,  etc.,  the  methods  consisted  in  the  employment  of  cultures 
of  the  living  organisms,  the  virulence  of  which  was  so  diminished 
that  on  inoculation  they  did  not  produce  a  fatal  disease,  but  yet 
had  effects  sufficient  for  protection.  The  principle  is  therefore 
the  same  as  that  of  vaccination,  and  the  attenuated  cultures  are 
often  spoken  of  as  vaccines.  The  virulence  of  an  organism  may 
be  diminished  in  various  ways,  of  which  the  following  examples 
may  be  given.  . 

(i)  In  the  first  place,  practically  every  organism,  when  culti- 
vated for  some  time  outside  the  body,  loses  its  virulence,  and 
in  the  case  of  some  this  is  very  marked  indeed,  e.g.  the  pneumo- 
coccus.  Pasteur  found  in  the  case  of  chicken  cholera,  that 


ACTIVE   IMMUNITY. 


465 


when  cultures  were  kept  for  a  long  time  in  ordinary  conditions, 
they  gradually  lost  their  virulence,  and  that  when  sub-cultures 
were  made,  the  diminished  virulence  persisted.  Such  attenuated 
cultures  could  be  used  for  protective  inoculation.  He  considered 
the  loss  of  virulence  to  be  due  to  the  action  of  the  oxygen  of  the 
air,  as  he  found  that  in  tubes  sealed  in  the  absence  of  oxygen 
the  virulence  was  not  lost.  Haffkine  attenuated  cultures  of  the 
cholera  spirillum  by  growing  them  in  a  current  of  air  (p.  421). 

(2)  The  virulence  of  an  organism  for  a  particular  animal  may 
be  lessened  by  passing  the  organism  through  the  body  of  another 
animal.     Duguid  and  Burdon  Sanderson  found  that  the  virulence 
of  the  anthrax  bacillus  for  bovine  animals  was  lessened  by  being 
passed  through  guinea-pigs,  the  disease  produced  in  the  ox  by 
inoculation  from  the  guinea-pig  being  a  non-fatal  one.     This  dis- 
covery was  confirmed  by  Greenfield,  who  found  that  the  bacilli 
cultivated  from  guinea-pigs  preserved  their  property  in  cultures, 
and  could  therefore  be  used 'for  protective  inoculation  of  cattle. 
A  similar  principle  was  applied  in  the  case  of  swine  plague  by 
Pasteur,  who  found  that  if  the  organism  producing  this  disease 
was  inoculated  from  rabbit  to  rabbit,  its  virulence  was  increased 
for  rabbits  but  was  diminished  for  pigs.     Organisms  which  had 
been  passed  through  a  series  of  rabbits  produced  in  the  pig 
illness,  but  not  death,  and  protection  for  at  least  a  year  resulted. 
The  method  of  vaccination  against  smallpox  depends  upon  the 
same  principle. 

(3)  Many  organisms  become  diminished  in  virulence  when 
grown  at  an  abnormally  high  temperature.     The  method  of 
Pasteur,  already  described  (p.  315),  for  producing  immunity  in 
sheep  against  anthrax  bacilli,  depends  upon  this  fact.    A  virulent 
organism  may  also  be  attenuated  by  being  exposed  to  an  elevated 
temperature  which  is  insufficient  to  kill  it.     Toussaint  at  an  early 
date  obtained  protective  inoculation  against  anthrax  by  means  of 
cultures  which  had  been  exposed  for  a  certain  time  to  a  tempera- 
ture of  55°  C,  though  it  is  possible  that  in  some  cases  the  bacilli 
were  really  killed,  and  immunity  resulted  from  the  chemical  sub- 
stances in  the  bacilli  or  produced  by  them. 

(4)  Still  another  method  may  be  mentioned,   namely,  the 
attenuation  of  the  virulence  by  growing  the  organism  in  the 
presence  of  weak  antiseptics.     Chamberland  and  Roux,  for  ex- 
ample, succeeded  in  attenuating  the  anthrax  bacillus  by  grow- 

2H 


466  IMMUNITY. 

ing  it  in  a  medium  containing  carbolic  acid  in  the  proportion 
of  i  :  600.  The  virulence  may  also  sometimes  be  attenuated 
by  injecting  certain  chemical  substances  along  with  the  bacteria 
into  the  body.  Iodine  terchloride  was  found  by  Behring  to 
modify  in  this  way  the  virulence  of  the  diphtheria  bacillus. 

These  examples  will  serve  to  show  the  principles  underlying 
attenuation  of  the  virulence  of  an  organism.  There  are,  how- 
ever, still  other  methods,  most  of  which  consist  in  growing  the 
organism  in  conditions  somewhat  unfavourable  to  its  growth,  e.g. 
under  compressed  air,  etc. 

(f)  By  living  Virulent  Cultures  in  Non-lethal  Doses.  —  Immu- 
nity may  also  be  produced  by  employing  virulent  cultures  in 
small,  that  is  non-lethal,  doses.  In  subsequent  inoculations  the 
doses  may  be  increased  in  amount.  For  example,  immunity 
may  thus  be  obtained  in  rabbits  against  the  bacillus  pyocyan- 
eus.  Such  a  method,  however,  has  had  a  limited  application  in 
the  case  of  virulent  organisms,  as  it  has  been  found  more  con- 
venient to  commence  the  process  by  attenuated  cultures. 

Exaltation  of  the  Virulence. — The  converse  process  to  attenua- 
tion, i.e.  the  exaltation  of  the  virulence,  is  obtained  chiefly  by  the 
method  of  cultivating  the  organism  from  animal  to  animal  —  the 
method  of  passage  discovered  by  Pasteur  (first,  we  believe,  in 
the  case  of  an  organism  obtained  from  the  saliva  in  hydro- 
phobia, though  having  no  causal  relationship  to  that  disease). 
This  is  most  conveniently  done  by  intraperitoneal  injections,  as 
there  is  less  risk  of  contamination.  The  organisms  in  the  peri- 
toneal fluid  may  be  used  for  the  subsequent  injection,  or  a  cul- 
ture may  be  made  between  each  inoculation.  The  virulence  of 
a  great  number  of  organisms  can  be  increased  in  this  way,  the 
animals  most  frequently  used  being  rabbits  and  guinea-pigs. 
This  method  can  be  applied  to  the  organisms  of  typhoid, 
cholera,  pneumonia,  to  streptococci,  and  staphylococci,  and  in 
fact  to  those  organisms  generally  which  invade  the  tissues. 

The  virulence  of  an  organism,  especially  when  in  a  relatively 
attenuated  condition,  can  also  be  raised  by  injecting  along  with 
it  a  quantity  of  a  culture  of  another  organism  either  in  the  living 
or  dead  condition.  A  few  examples  may  be  mentioned.  An 
attenuated  diphtheria  culture  may  have  its  virulence  raised  by 
being  injected  into  an  animal  along  with  the  streptococcus 
pyogenes ;  an  attenuated  culture  of  the  bacillus  of  malignant 


BY   BACTERIAL   PRODUCTS   OR   TOXINS.  467 

oedema  by  being  injected  with  the  bacillus  prodigiosus ;  an 
attenuated  streptococcus  by  being  injected  with  the  bacillus  coli, 
etc.  A  culture  of  the  typhoid  bacillus  may  be  increased  in 
virulence,  as  already  stated,  by  being  injected  along  with  a  dead 
culture  of  the  bacillus  coli.  In  such  cases  the  accompanying 
injection  enables  the  attenuated  organism  to  gain  a  foothold  in 
the  tissues,  and  it  may  be  stated  as  a  general  rule  that  the 
virulence  of  an  organism  for  a  particular  animal  is  raised  by  its 
growing  in  the  tissues  of  that  animal. 

Combination  of  Methods.  —  The  above  methods  may  be  com- 
bined in  various  ways.  By  repeated  injections  of  cultures  at  first 
attenuated  and  afterwards  more  virulent,  and  by  increasing  the 
doses,  a  high  degree  of  immunity  may  be  obtained.  This  is 
well  exemplified  in  the  case  of  Haffkine's  method  of  anti-cholera 
inoculation  (p.  421). 

2.  Immunity  by  Dead  Cultures  of  Bacteria.  —  In  some  cases 
a  high  degree  of  immunity  against  infection  by  a  given  microbe 
may  be  developed  by  repeated  and  gradually  increasing  doses 
of  the  dead  cultures,  the  cultures  being  killed  sometimes  by 
heat,  sometimes  by  exposure  to  the  vapour  of  chloroform.    Some 
consider  that  in  this  method  only  the  intracellular  toxic  sub- 
stances of  the  organism  are  introduced  when  the  cultures  have 
been  taken  from  the  surface  of  a  solid  medium,  such  as  agar,  but 
as  the  surface  is  moist,  some  of  the  extracellular  products  must 
be  present  also.    The  cultures  when  dead  produce,  of  course,  less 
effect  than  when  living,  and  this  method  may  be  conveniently 
used  in  the  initial  stages  of  active  immunisation,  to  be  afterwards 
followed  by  injections  of  the  living  cultures.     The  method  is 
extensively  used  for  experimental  purposes,  and  is  that  adopted 
in  anti-plague  and  anti-typhoid  inoculations. 

3.  Immunity  by  the  Separated  Bacterial  Products  or  Toxins. 
—  The  organisms  in  a  virulent  condition  are  grown  in  a  fluid 
medium  for  a  certain  time,  and  the  fluid  is  then  filtered  through 
a  Chamberland  or  other  porcelain  filter.     The  filtrate  contains 
the  toxins,  and  it  may  be  used  unaltered,  or  may  be  reduced  in 
bulk  by  evaporation,  or  may  be  evaporated  to  dryness.     The 
process  of  immunisation  by  the  toxin  is  started  by  small,  non- 
lethal  doses  of  the  strong  toxin,  or  by  larger  doses  of  toxin  the 
power  of  which  has  been  weakened  by  various  methods  (vide 
infra).      Afterwards  the   doses   are   gradually  increased.      Im- 


468  IMMUNITY. 

munity  produced  in  this  way  is  effective  not  only  against  the 
toxin,  but  also  against  large  doses  of  the  virulent  organism  in  a 
living  condition.  This  method  was  carried  out  with  a  great 
degree  of  success  in  the  case  of  diphtheria,  tetanus,  malignant 
oedema,  etc.  It  appears  capable  of  very  general  application, 
though,  in  the  case  of  many  organisms,  it  is  difficult  to  get  a 
very  active  toxin  from  the  filtered  cultures.  It  has  also  been 
applied  in  the  case  of  snake  poisons  by  Calmette  and  Fraser, 
and  a  high  degree  of  immunity  has  been  produced. 

Immunity  may  also  be  obtained  by  means  of  certain  chemical 
substances  separated  from  filtered  bacterial  cultures,  though 
these  substances  are  generally  in  a  more  or  less  impure  condition. 
Hankin  was  the  first  to  obtain  this  result  by  means  of  an 
^albumose  separated  from  anthrax  cultures. 

Though,  as  already  stated,  none  of  these  methods  can  be 
used  directly  as  curative  agents,  seeing  that  they  imply  previous 
treatment  before  exposure  to  infection,  yet  they  supply  the 
means  of  developing  a  very  high  degree  of  immunity,  which 
is  the  first  stage  in  the  production  of  an  active  curative  serum. 

The  following  may  be  mentioned  as  some  of  the  most 
important  examples  of  the  practical  application  of  the  principles 
of  active  immunity,  i.e.  of  protective  inoculation:  (i)  Inocula- 
tion of  sheep  and  oxen  against  anthrax  (Pasteur)  (p.  315);  (2) 
Jennerian  vaccination  against  smallpox  (p.  501);  (3)  Anti-cholera 
inoculation  (Haffkine)  (p.  421);  (4)  Anti-plague  inoculation 
(Haffkine)  (p.  444);  (5)  Anti-typhoid  inoculation  (Wright  and 
Semple  (p.  346) ;  (6)  Pasteur's  method  of  inoculation  against 
hydrophobia,  which  involves  essentially  the  same  principles. 

Active  Immunity  by  Feeding.  —  Ehrlich  found  that  mice 
could  be  gradually  immunised  against  ricin  and  abrin  by  feeding 
them  with  increasing  quantities  of  these  substances  (vide  p.  177). 
In  the  course  of  some  weeks'  treatment  in  this  way  the  resulting 
immunity  was  of  so  high  a  degree  that  the  animals  could  tolerate 
400  times  the  dose  originally  fatal  by  subcutaneous  inoculation. 
Fraser  also  found  in  the  case  of  snake  poison  that  rabbits  could 
be  immunised,  by  feeding  with  the  poisons,  against  several  times 
the  lethal  dose  of  venom  injected  into  the  tissues. 

By  feeding  animals  with  dead  cultures  of  bacteria  or  with 
their  separated  toxins,  a  certain  degree  of  immunity  may  in 
certain  cases  be  gradually  developed.  But  this  method  is  so 


PASSIVE   IMMUNITY.  469 

much  less  certain  in  results,  and  so  much  more  tedious  than  the 
others,  that  it  has  obtained  no  practical  applications. 

Active  immunity  of  high  degree  developed  by  the  methods 
described  may  be  regarded  as  specific,  that  is,  is  exerted  only 
toward  the  organism  or  toxin,  by  means  of  which  it  has  been 
produced.  A  certain  degree  of  immunity,  or  rather  of  increased 
general  resistance  of  parts  of  the  body  (for  example  the  peri- 
toneum), can,  however,  be  produced  by  the  injection  of  various 
substances  —  bouillon,  blood  serum,  solution  of  nuclein,  etc. 
(Issaeff).  Also  increased  resistance  to  one  organism  can  be 
thus  produced  by  injections  of  another  organism.  Immunity  of 
this  kind,  however,  never  reaches  a  high  degree. 

\  B.    Passive  Immunity. 

Action  of  the  Serum  of  highly  Immunised  Animals.  —  i.  The. 
serum  of  an  animal  A,  treated  by  repeated  and  gradually  in-> 
creased  doses  of  the  toxin  of  a  particular  microbe,  may  protect 
an  animal  B  against  a  certain  amount  of  the  same  toxin  when 
injected  along  with  the  latter,  or  a  short  time  before  it.     As 
would  be  expected,  it  has  less  effect  when  injected  some  time 
afterwards,  but  even  then  within  certain  limits  it  has  a  degree  of 
curative  or  palliative  power.     Seeing  that  the  serum  of  animal 
A  appears  to  neutralise  the  toxin,  the  term  antitoxic  has  been 
applied  to  it. 

2.  The  serum  of  an  animal  A,  highly  immunised  against  a 
microbe  by  repeated  and  gradually  increasing  doses  of  the  living 
organism,  protects  an  animal  B  against  an  infection  by  the  living 
organism  when  injected  under  conditions  similar  to  the  above. 
This  serum  is  therefore  antimicrobic,  or  antibacterial,  or  pre- 
ventive against  invasion  by  a  particular  organism.  When  spoken 
of  in  relation  to  the  bacterium  by  means  of  which  it  has  been 
prepared,  a  serum  is  usually  called  homologous ;  in  relation  to 
any  other  bacterium,  heterologoits. 

In  a  considerable  number  of  instances,  an  antimicrobic  serum 
has  been  found  to  possess  little  effect  against  the  toxin  —  that 
is,  to  possess  little  or  no  antitoxic  power.  This  fact,  if  taken 
alone,  would  leave  it  still  doubtful  whether  the  difference  between 
the  two  kinds  of  sera  were  one  of  quality  or  one  merely  of  quan- 
tity. It  has,  however,  been  shown  in  many  cases  that  an  anti- 
microbic serum  has  a  distinct  action  on  the  vital  activity  of  the 


4/0  IMMUNITY. 

corresponding  bacterium,  and  may  even  produce  alteration  in 
its  structure.  It  is  manifest  that  such  a  serum  differs  funda- 
mentally in  its  point  of  attack,  so  to  speak,  from  an  antitoxic 
serum.  And  it  is  to  be  noted  that  the  nature  of  the  serum 
corresponds  in  a  wonderful  way  with  the  requirements  of  the 
organism,  that  is,  is  antitoxic  in  such  diseases  as  diphtheria  and 
tetanus  where  toxic  action  is  at  its  maximum,  and  bactericidal 
where  the  rapid  multiplication  of  the  bacteria  in  the  tissues  is 
the  outstanding  feature.  It  must  not  be  supposed,  however, 
that  a  serum  must  be  purely  antitoxic  or  purely  antibacterial 
according  to  the  method  by  which  it  is  prepared.  For  example, 
an  antitoxic  serum  can  be  obtained  by  injecting  living  diphtheria 
bacilli  into  the  tissues  of  an  animal,  the  antitoxic  property  being 
in  all  probability  developed  by  means  of  toxins  formed  by  the 
bacilli  within  the  body.  Having  given  this  explanation,  we  shall 
consider  the  two  kinds  of  serum  separately. 

Antitoxic  Serum.  —  The  best  examples  are  the  antitoxic 
sera  of  diphtheria  and  tetanus,  though  similar  principles  and 
methods  are  involved  in  the  preparation  of  the  sera  protective 
against  ricin  and  abrin,  and  against  snake  poison.  We  shall 
here  speak  of  diphtheria  and  tetanus.  The  steps  in  the  process 
of  preparation  may  be  said  to  be  the  following  :  First,  the  prepa- 
ration of  a  powerful  toxin.  Second,  the  estimation  of  the  power 
of  the  toxin.  Third,  the  development  of  antitoxin  in  the  blood 
of  a  suitable  animal  by  gradually  increasing  doses  of  the  toxin. 
Fourth,  the  estimation  from  time  to  time  of  the  antitoxic  power 
of  the  serum  of  the  animal  thus  treated. 

1.  Preparation  of  the  Toxin.  — The  mode  of  preparation  and 
the   conditions   affecting  the   development  of  diphtheria  toxin 
have  already  been  described  (p.  366).     In  the  case  of  tetanus 
the  growth  takes  place  in  glucose  bouillon  under  an  atmosphere 
of  hydrogen  (vide  p.  63).     In  either  case  the  culture  is  filtered 
through  a  Chamberland   filter  when    the   maximum  degree  of 
toxicity  has  been  reached.     The  term  "  toxin  "  is  usually  applied 
for  convenience  to  the  filtered  (i.e.  bacterium-free)  culture. 

2.  Estimation  of  the  Toxin.  —  The  power  of  the  toxin  is  es- 
timated by  the  subcutaneous  injection  of  varying  amounts  in  a 
number  of  guinea-pigs,  and  the  minimum  dose  which  will  produce 
death  is  thus  obtained.     This,  of  course,  varies  in  proportion  to 
the  weight  of  the  animal,  and  is  expressed  accordingly.     In  the 


DEVELOPMENT   OF  ANTITOXIN.  471 

case  of  diphtheria,  in  Ehrlich's  standard,  the  minimum  lethal 
dose  —  known  as  M.L.D.  — is  the  smallest  amount  which  will 
certainly  cause  death  in  a  guinea-pig  of  250  grms.  within  four 
days.  Behring  uses  the  term  "  normal  diphtheria  toxin  of  simple 
strength"  (DTN1),  as  indicating  a  toxin  of  which  .01  c.c.  is  the 
minimum  lethal  dose  under  these  conditions.  A  toxin  of  which 
the  minimum  lethal  dose  is  .02  will  be  of  half  normal  strength 
(DTN-5) ;  and  so  on.  The  testing  of  a  toxin  directly  is  a  tedious 
process,  and  in  actual  practice,  where  many  toxins  have  to  be 
dealt  with,  it  is  found  more  convenient  to  test  them  by  finding 
how  much  will  be  neutralised  by  a  certain  amount  of  a  standard 
antitoxic  serum,  viz.,  an  "immunity  unit"  (p.  472). 

3.  Development  of  Antitoxin.  —  The  earlier  experiments  on 
tetanus  and  diphtheria  were  performed  on  the  small  animals, 
such  as  guinea-pigs,  but  afterwards  the  sheep  and  the  goat  were 
used,  and  finally  horses.  In  the  case  of  the  small  animals  it 
was  found  advisable  to  use  in  the  first  stages  of  the  process 
either  a  weak  toxin  or  a  powerful  toxin  modified  by  certain 
methods.  Such  methods  are  the  addition  to  the  toxin  of  ter- 
chloride  of  iodine  (Behring  and  Kitasato),  the  addition  of  Gram's 
iodine  solution  in  the  proportion  of  one  to  three  (Roux  and 
Vaillard),  and  the  plan,  adopted  by  Vaillard  in  the  case  of 
tetanus,  of  using  a  series  of  toxins  weakened  to  varying  degrees 
by  being  exposed  to  different  temperatures,  viz.,  60°,  55°,  and 
50°  C.  The  toxin  is  at  first  injected  into  the  subcutaneous 
tissues,  the  dose  being  gradually  increased  according  to  the 
results  of  the  toxin  injected.  As  pointed  out  by  Behring,  im- 
munisation proceeds  best  when  each  injection  produces  a  reac- 
tion in  the  form  of  localised  inflammatory  swelling ;  in  other 
words,  the  dose  should  be  as  large  as  possible,  so  long  as  gen- 
eral injurious  effects  are  not  produced.  Later,  when  large  doses 
of  toxin  injected  subcutaneously  are  well  borne,  the  toxin  is 
injected  directly  into  the  jugular  vein  of  the  animal.  Ultimately 
300  c.c.,  or  more,  of  active  diphtheria  toxin  thus  injected  may 
be  borne  by  a  horse,  such  a  degree  of  resistance  being  developed 
after  the.  treatment  has  been  carried  out  for  two  or  three  months. 
In  all  cases  of  immunising  the  general  health  of  the  animal 
ought  not  to  suffer.  If  the  process  is  pushed  too  rapidly  the 
antitoxic  power  of  the  serum  may  diminish  instead  of  increasing, 
and  a  condition  of  marasmus  may  set  in  and  may  even  lead  to 


4/2  IMMUNITY. 

the  death  of  the  animal.  (In  immunisation  of  small  animals  an 
indication  of  their  general  condition  may  be  obtained  by  weigh- 
ing them  from  time  to  time.) 

4.  Estimating  the  Antitoxic  Power  of,or"  standardising"  tJie 
Serum.  —  This  is  done  by  testing  the  effect  of  various  quantities 
of  the  serum  of  the  immunised  animal  against  a  certain  amount 
of  toxin.  Various  standards  have  been  used,  of  which  the  two 
chief  are  that  of  Ehrlich  and  that  of  Roux.  Ehrlich  has  adopted 
as  the  immunity  unit  the  amount  of  antitoxic  serum  which  will 
neutralise  100  times  the  minimum  lethal  dose  of  toxin,  serum 
and  toxin  being  mixed  together,  diluted  up  to  4  c.c.  and  injected 
subcutaneously.  A  "normal"  antitoxic  serum  is  one  of  which 
i  c.c.  contains  an  immunity  unit.  Owing  to  the  difficulty  of 
estimating  the  occurrence  of  local  infiltration  at  the  site  of 
injection,  the  prevention  of  the  death  of  the  animal  within  four 
days  is  used  as  the  sole  indication  of  neutralisation,  —  death 
later  or  loss  of  weight  and  local  infiltration  being  neglected.  As 
a  standard  in  testing,  Ehrlich  employs  quantities  of  serum  of 
known  antitoxic  power  in  a  dry  condition,  preserved  in  a  vacuum 
in  a  cool  place,  and  in  the  absence  of  light.  A  thoroughly  dry 
condition  is  ensured  by  having  the  glass  bulb  containing  the 
dried  serum  connected  with  another  bulb  containing  anhydrous 
phosphoric  acid.  Thus  I  c.c.  of  a  serum  of  which  .02  c.c.  will 
protect  from  100  times  the  lethal  dose,  will  possess  50  immunity 
units,  and  20  c.c.  of  this  serum  1000  immunity  units.  Sera  have 
been  prepared  of  which  I  c.c.  has  the  value  of  800  units  or  even 
more. 

Roux  adopts  a  standard  which  represents  the  animal  weight  in  grammes 
protected  by  i  c.c.  of  serum  against  the  dose  of  virulent  bacilli  lethal  to  a 
control  guinea-pig  in  thirty  hours,  the  serum  being  injected  twelve  hours 
previously.  Thus,  if  .01  c.c.  of  a  serum  will  protect  a  guinea-pig  of  500 
gratis,  against  the  lethal  dose,  i  c.c.  (i  grm.)  will  protect  50,000  grms.  of 
guinea-pig,  and  the  value  of  the  serum  will  be  50,000. 

During  the  process  of  development  of  antitoxin  a  small 
quantity  of  the  blood  of  the  animal  is  withdrawn  from  time  to 
time,  and  the  antitoxic  power  tested  in  the  manner  described 
above.  After  a  sufficiently  high  degree  of  antitoxic  power  has 
been  reached  the  animal  is  bled  under  aseptic  precautions,  and 
the  serum  is  allowed  to  separate  in  the  usual  manner.  It  is  then 
ready  for  use,  but  some  weak  antiseptic,  such  as  .5  per  cent 


DOSAGE   OF   DIPHTHERIA   ANTITOXIN.  473 

carbolic  acid,  is  usually  added  to  prevent  its  decomposing.  Other 
antitoxic  sera  are  prepared  in  a  corresponding  manner.  Some 
further  facts  about  antitetanic  serum  are  given  on  page  389. 

Use  of  Antitoxic  Sera.  —  In  all  cases  the  antitoxic  serum 
ought  to  be  injected  as  early  in  the  disease  as  possible,  and  in 
large  doses.  In  the  case  of  diphtheria  1500  immunity  units  of 
antitoxic  serum  was  the  amount  first  recommended  for  the  treat- 
ment of  a  bad  case,  but  the  advisability  of  using  larger  doses 
has  gradually  become  more  and  more  evident.  Sidney  Martin 
recommends  that  as  much  as  4000  units  should  be  administered 
at  once,  and  that  if  necessary  this  quantity  should  be  repeated. 
A  strong  serum  prepared  by  Behring  contains  3000  units  in 
5-6  c.c.,  but  even  stronger  serum  may  be  obtained.  Even  very 
large  doses  of  antitoxic  serum  are  without  any  harmful  effects 
beyond  the  occasional  production  of  urticarial  and  erythematous 
rashes.  Where  large  quantities  of  serum  require  to  be  admin- 
istered, as  is  always  the  case  with  antitetanic  serum,  injections 
must  be  made  at  different  parts  of  the  body ;  preferably  not 
more  than  20  c.c.  should  be  injected  at  one  place.  The  immu- 
nity conferred  by  injection  of  antitoxic  serum  lasts  a  compara- 
tively short  time,  usually  a  few  weeks  at  longest. 

Sera  of  Animals  immunised  against  Vegetable  and  Animal 
Poisons.  —  It  was  found  by  Ehrlich  in  the  case  of  the  vegetable 
toxins,  ricin  and  abrin,  and  also  by  Calmette  and  Fraser  in  the 
case  of  the  snake  poisons,  that  the  serum  of  animals  immunised 
against  these  respective  substances  had  a  protective  effect  when 
injected  along  with  them  into  other  animals.  Ehrlich  found, 
for  example,  that  the  serum  of  a  mouse  which  had  been  highly 
immunised  against  ricin  by  feeding  as  described  above,  could 
protect  another  mouse  against  forty  times  the  fatal  dose  of  that 
substance.  He  considered  that  in  the  case  of  the  two  poisons, 
antagonistic  substances  —  "  anti-ricin"  and  "  anti-abrin  "  —  were 
developed  in  the  blood  of  the  highly  immunised  animals.  A 
corresponding  antagonistic  body,  to  which  Fraser  has  given  the 
name  "  antivenin,"  appears  in  the  blood  of  animals  in  the  process 
of  immunisation  against  snake  poison. 

These  investigations  are  specially  instructive,  as  these  vege- 
table and  animal  poisons, -both  as  regards  their  local  action  and 
the  general  toxic  phenomena  produced  by  them,  present,  as  we 
have  seen,  an  analogy  to  various  toxins  of  bacteria. 


474  IMMUNITY. 

Nature  of  Antitoxic  Action. — We  have  to  consider  here  two 
points,  viz.,  (a)  the  relation  of  antitoxin  to  toxin,  and  (U)  the 
source  of  the  antitoxin.  With  regard  to  the  former  subject  there 
has  been  much  diversity  of  opinion.  Some  observers  consider 
that  the  antagonism  between  toxin  and  antitoxin  depends  upon 
a  chemical  union  between  the  two  substances,  whilst  others  con- 
sider that  it  is  of  a  physiological  nature,  the  antitoxin  acting 
through  the  medium  of  the  cells  of  the  organism.  The  bulk  of 
evidence  recently  brought  forward  is,  however,  strongly  in  favour 
of  the  view  that  the  two  bodies  unite  in  vitro  to  form  a  compound 
inert  towards  the  living  tissues,  there  being  in  the  toxin  molecule 
an  atom-group  which  has  a  specific  affinity  for  the  antitoxin 
molecule  or  part  of  it.  We  shall  consider  the  facts  in  favour  of 
this  view,  and  in  doing  so  we  must  also  take  into  account  the 
anti-sera  of  the  vegetable  toxins,  of  snake  poisons,  etc. 

When  toxin  and  antitoxin  are  brought  together  in  vitro  it 
can  be  proved  that  their  behaviour  towards  each  other  resembles 
what  is  observed  in  a  simple  chemical  union.  It  is  of  course  to 
be  kept  in  view  that  the  only  test  of  the  neutralisation  of  the 
toxin  by  the  antitoxin  is  that  when  the  resultant  mixture  is 
injected  into  a  susceptible  animal  no  symptoms  occur.  As  in 
chemical  union,  a  definite  period  of  time  elapses  before  the 
neutralisation  of  the  toxin  is  complete.  Other  points  of  resem- 
blance to  simple  chemical  union  are  found  in  the  facts  that 
neutralisation  takes  place  more  rapidly  in  strong  solutions  than 
in  weak,  and  that  it  is  hastened  by  warmth  and  delayed  by  cold. 
It  has  been  found  that  if  these  factors  be  taken  into  account 
and  a  standard  toxin  of  definite  strength  be  employed,  a  toxin 
can  be  titrated  against  an  antitoxin  with  corresponding  accuracy 
to  what  obtains  in  the  case  of  an  acid  and  an  alkali.  C.  J. 
Martin  and  Cherry  and  also  Brodie  have  shown  that  in  the  case 
of  diphtheria  toxin  and  in  that  of  an  Australian  snake  poison 
the  toxin  molecules  will  pass  through  a  colloid  membrane  (p.  1/4), 
whilst  those  of  the  corresponding  antitoxin  will  not.  Now  if  a 
mixture  of  equivalent  parts  of  toxin  and  antitoxin  is  freshly 
prepared  and  at  once  filtered,  a  certain  amount  of  toxin  will  pass 
through,  but  the  longer  such  mixtures  are  allowed  to  stand  before 
filtration  the  less  toxin  passes,  till  a  time  is  reached  when  no 
toxin  is  found  in  the  filtrate.  Further,  if  the  portion  of  fluid 
which  at  this  stage  has  not  passed  through  the  filter  be  injected 


NATURE   OF   ANTITOXIC   ACTION. 


475 


into  an  animal,  no  symptoms  take  place.  This  shows  that  after 
a  time  neutralisation  is  complete.  These  facts  are  practically 
conclusive  in  favour  of  toxin  neutralisation  depending  upon  a 
chemical  union,  and  such  a  view  would  also  throw  light  on  the 
otherwise  somewhat  puzzling  fact  that  while,  e.g.  by  lapse  of 
time,  the  toxicity  of  a  toxin  may  become  diminished,  it  may  still 
require  the  same  proportion  of  antitoxin  to  neutralise  it  as  it  did 
before.  On  the  chemical  theory  this,  according  to  Ehrlich,  is 
due  to  the  disintegration  of  the  toxophorous  atom-group  of  the 
toxin  molecule  (vide  pp.  179,  478),  while  the  combining  (hapto- 
phorous)  group  still  remains  unaltered.  Quite  analogous  cases 
could  be  cited  from  pure  chemistry. 

The  evidence  usually  brought  forward  against  the  chemical  union  of  toxin 
and  antitoxin  rests  chiefly  on  certain  observations  of  Buchner  and  of  Calmette, 
and  to  these  a  reference  must  be  made.  Buchner,  in  a  series  of  experiments, 
came  to  the  conclusion  that  it  was  possible  to  make  a  mixture  of  tetanus  toxin 
and  antitoxin  which  was  neutral  to  the  mouse,  but  which  could  produce  a  fatal 
result  in  guinea-pigs.  It  is  to  be  noted,  however,  that  the  mixture  used  in  his 
experiments  was  not  quite  neutral  to  mice,  and  this  circumstance,  along  with 
the  fact  that  the  guinea-pig,  weight  for  weight,  is  more  susceptible  to  this  toxin 
than  the  mouse,  may  explain  the  result.  In  any  case  these  experiments  as 
they  stand  cannot  be  considered  to  constitute  a  real  objection.  Calmette  found 
that  the  antitoxin  to  snake  venom  was  more  easily  destroyed  by  heat  than  the 
toxin,  and  stated  that  when  a  neutral  mixture  of  the  two  was  heated  at  a 
temperature  sufficient  to  destroy  free  antivenin,  the  toxic  properties  in  part 
returned.  Hence  he  concluded  that  the  two  bodies  existed  in  an  uncombined 
condition  in  the  mixture.  Martin  and  Cherry,  however,  on  repeating  these 
experiments,  found  that  the  above  result  was  not  obtained  if  sufficient  time  for 
complete  combination  was  allowed ;  but  if  this  precaution  was  not  taken,  then 
the  presence  of  the  free  toxin  was  revealed  when  the  antitoxin  was  destroyed 
by  heat.  Even,  however,  if  Calmette's  results  were  quite  correct,  they  cannot 
be  considered  t(5  constitute  a  proof  that  chemical  union  does  not  occur :  they 
would  only  prove  that  the  toxin  has  not  been  destroyed.  If  two  complicated 
chemical  compounds  of  unequal  stability  are  in  loose  chemical  union,  it  is 
quite  conceivable  that  the  less  stable  may  be  destroyed  (e.g.  by  heat)  whilst 
the  more  stable  escapes. 

The  next  question  to  be  considered  is  the  source  of  antitoxin. 
The  following  three  possibilities  present  themselves  :  (a)  antitoxin 
may  be  formed  from  the  toxin,  i.e.  may  be  a  "  modified  toxin  "  ; 
(b)  antitoxin  may  be  the  result  of  an  increased  formation  of 
molecules  normally  present  in  the  tissues ;  (c)  antitoxin  may  be 
an  entirely  new  product  of  the  cells  of  the  body.  It  can  now  be 
stated  that  antitoxin  is  not  a  modified  toxin.  It  has  been  shown, 


476  IMMUNITY. 

for  example,  that  the  amount  of  antitoxin  produced  by  an  animal 
may  be  many  times  greater  than  the  equivalent  of  toxin  injected  ; 
and  further,  that  when  an  animal  is  bled  the  total  amount  of 
antitoxin  in  the  blood  may  some  time  afterwards  be  greater  than 
it  was  immediately  after  the  bleeding,  even  although  no  addi- 
tional toxin  is  introduced.  This  latter  circumstance  shows 
antitoxin  is  formed  by  the  cells  of  the  body.  If  antitoxin  is  a 
product  of  the  cells  of  the  body,  we  are  almost  compelled,  on 
theoretical  grounds,  to  conclude  that  it  is  not  a  newly  manufac- 
tured substance,  but  a  normal  constituent  of  the  living  cells 
which  is  produced  in  increased  quantity.  We  have,  however, 
direct  evidence  of  the  presence  of  antitoxin  under  normal  con- 
ditions, —  the  presence  of  such  being  shown  by  its  uniting  with 
toxin  and  rendering  it  inert.  Normal  horse  serum,  to  mention 
an  example,  may  have  a  varying  amount  of  antitoxic  action  to 
the  diphtheria  poison,  ox-bile  has  a  similar  action  to  snake  poison, 
whilst  in  the  case  of  other  anti-substances  —  such  as  agglutinins, 
bacteriolysins,  haemolysins,  etc.  — whose  production  is  governed 
by  the  same  laws,  numerous  examples  might  be  given.  It  is, 
however,  rather  to  the  protoplasm  of  living  cells  than  to  the  serum 
that  we  must  look  for  evidence  of  antitoxins.  In  the  first  place, 
we  have  evidence  that  in  the  living  body  bacterial  toxins  enter 
into  combination  with,  or,  as  it  is  often  expressed,  are  fixed  by 
the  tissues —  presumably  by  means  of  certain  combining  affinities. 
This  has  been  shown  by  the  experiments  of  Donitz  and  of  Hey- 
mans  with  tetanus  toxin.  We  have,  however,  no  evidence  as  to 
where  the  toxin  is  fixed  in  such  cases  beyond  that  supplied  by  the 
occurrence  of  symptoms.  Another  line  of  research  which  has 
been  followed  is  to  bring  emulsions  of  various  organs  into  con- 
tact with  a  given  toxin  and  observe  whether  any  of  the  toxicity 
is  removed.  This  was  first  carried  out  by  Wassermann  and 
Takaki,  who  investigated  the  action  of  emulsions  of  the  central 
nervous  system  of  the  susceptible  guinea-pig  on  tetanus  toxin. 
They  found  in  this  way  that  the  nervous  system  contained  bodies 
which  had  a  neutralising  effect  on  the  toxin.  For  example,  it 
was  shown  that  I  c.c.  of  emulsion  of  brain  and  spinal  cord  was 
capable  of  protecting  a  mouse  against  ten  times  the  fatal  dose 
of  toxin.  These  observations  have  been  confirmed,  though  their 
significance  has  been  variously  interpreted.  It  would,  however, 
be  out  of  place  to  discuss  at  length  the  opposing  views,  and  we 


CHEMICAL   NATURE   OF   ANTITOXINS. 


477 


accordingly  simply  state  the  facts  ascertained.  We  may  note, 
however,  that  it  is  not  a  serious  objection  that  in  certain  animals 
other  tissues  than  that  of  the  central  nervous  system  can  combine 
with  tetanus  toxin  — this  might  take  place  with  or  without  result- 
ing symptoms ;  the  important  fact  is  that  in  the  nervous  system 
certain  molecules  have  an  affinity  for  the  toxin. 

It  will  be  seen  from  what  has  been  stated  with  regard  to  the 
relation  of  toxin  and  antitoxin,  that  the  fixation  of  toxin  by  the 
tissues  leads  up  theoretically  to  the  possible  production  of  anti- 
toxin. In  other  words,  the  substance  which,  when  forming  part 
of  the  cells,  fixes  the  toxin  and  thus  serves  as  the  means  of 
poisoning,  may  act  as  an  antitoxin  when  free  in  the  blood.  This 
will  be  discussed  below  in  connection  with  Ehrlich's  theory  of 
passive  immunity. 

We  may  conclude  this  portion  of  the  subject  by  saying  that 
(i)  it  is  practically  proved  that  antitoxin  acts  as  such  by  combining 
with  toxin,  and  (2)  antitoxin  is  probably  represented  by  'molecules 
normally  present  in  the  cells  or  (more  rarely)  in  the  fluids  of  the 
body. 

Within  recent  years  a  large  number  of  anti-substances  have 
been  obtained  against  substances  other  than  toxins.  As  ex- 
amples we  may  mention  precipitins,  which  are  produced  by  the 
injection  of  the  serum  of  another  animal,  and  which  produce 
an  opacity  when  added  to  that  serum  ;  various  anti-ferments ',  e.g. 
anti-rennet,  anti-coagulins,  also  anti-complements  (vide  p.  482). 
All  these  act  in  a  manner  corresponding  to  antitoxins ;  for 
instance,  the  addition  of  anti-rennet  to  milk  prevents  the  latter 
being  curdled  by  rennet.  Their  production  is  apparently 
governed  by  the  same  laws. 

Of  the  chemical  nature  of  antitoxins  we  know  little.  From 
their  experiments  C.  J.  Martin  and  Cherry  deduce  that  while 
toxins  are  probably  of  the  nature  of  albumoses,  the  antitoxins 
probably  have  a  molecule  of  greater  size,  and  may  be  allied  to 
the  globulins.  Hiss  and  Atkinson  have  also  come  to  the  con- 
clusion that  antitoxin  belongs  to  the  globulins.  They  found  that 
the  precipitate  with  magnesium  sulphate  from  anti-diphtheria 
serum  contained  practically  all  the  antitoxin,  and  that  any  sub- 
stance obtained  which  had  an  antitoxic  value  gave  all  the 
reactions  of  a  globulin.  They  also  found  that  the  percentage 
amount  of  globulin  precipitated  from  the  serum  of  the  horse 


478  IMMUNITY. 

increased  after  it  was  treated  in  the  usual  way  for  the  production 
of  antitoxin.  Such  a  supposed  difference  in  the  sizes  of  the 
molecules  might  explain  the  fact,  observed  by  Eraser  and  also 
by  C.  J.  Martin,  that  antitoxin  is  much  more  slowly  absorbed 
when  introduced  subcutaneously  than  is  the  case  with  toxin. 

Antitoxin,  when  present  in  the  serum,  leaves  the  body  by 
the  various  secretions,  and  in  these  it  has  been  found,  though 
in  much  less  concentration  than  in  the  blood.  It  is  present  in 
the  milk,  and  a  certain  degree  of  immunity  can  be  conferred  on 
animals  by  feeding  them  with  such  milk,  as  has  been  shown  by 
Ehrlich,  Klemperer,  and  others.  Klemperer  also  found  traces 
of  antitoxin  in  the  yolk  of  eggs  of  hens  whose  serum  contained 
antitoxin.  Bulloch  also  found  in  the  case  of  haemolytic  sera 
(vide  infra)  that  the  anti-substance  ("immune-body")  is  trans- 
mitted from  the  mother  to  the  offspring. 

The  Evidence  for  Ehrlich's  Theory  of  the  Constitution  of  Toxins.  —  This 
was  found  in  the  course  of  investigations  on  diphtheria  toxin,  and  so  far 
applies  only  to  the  extracellular  toxins.  Ehrlich  found  that,  taking  an  anti- 
toxin standardised  against  one  toxin,  it  did  not  follow  that  it  would  neutralise 
exactly  the  same  number  of  M.L.D.  of  other  toxins.  Thus  an  amount  which 
would  neutralise  100  M.L.D.  of  one,  might  neutralise  only  20  of  another  and 
perhaps  130  of  a  third.  The  second  fundamental  observation  was  as  follows  : 
If  a  mixture  of  toxin  and  antitoxin  behaved  like  a  mixture  of  say  hydrochloric 
acid  and  sodium  hydrate,  then  the  addition  to  a  neutral  mixture  of  i  M.L.D. 
would,  if  the  mixture  were  injected  into  a  guinea-pig,  cause  death.  In  none 
of  the  toxins  investigated  was  this  the  case  ;  sometimes  as  many  as  28  M.L.D. 
had  to  be  thus  added  before  death  occurred.  A  third  fact  observed  was  that 
in  the  case  of  one  toxin  when  freshly  filtered  the  M.L.D.  was  found  to  be 
.003  c.c. ;  nine  months  later  it  was  .009  c.c.,  but  it  was  found  that  after  the 
lapse  of  this  period  one  antitoxin  unit  neutralised  exactly  the  same  amount  of 
toxin  as  at  first.  In  other  words,  one  antitoxin  unit  when  the  toxin  was  fresh 
neutralised  100.2  M.L.D.,  and  nine  months  later  only  33.4  M.L.D.,  and  care 
had  been  taken  that  the  antitoxin  itself  had  not  changed.  The  theory  to 
account  for  these  facts  is  that  the  ultimate  toxin  molecule  contains  two  un- 
satisfied affinities,  one  of  which  can  combine  with  antitoxin,  the  other  having 
a  toxic  action ;  the  former  Ehrlich  calls  the  "  haptophorous  "  group,  the  latter 
the  "  toxophorous  "  (vide  p.  179).  Further,  each  of  these  groups  can,  under 
the  action  of  light,  oxidation,  etc.,  lose  a  certain  amount  of  combining  power, 
the  toxophorous  being  more  easily  weakened  than  the  haptophorous  (these 
weakened  toxins  Ehrlich  calls  "  toxoids  "  or  "toxones").  Now,  the  above 
facts  can  be  explained  if  crude  diphtheria  toxin  contains  both  of  these  sub- 
stances, i.e.  true  toxin  with  powerful  haptophorous  and  toxophorous  affinities, 
and  toxoid  with  slightly  weakened  haptophorous  group  and  greatly  weakened 
toxophorous  group.  Take  the  second  fundamental  observation  alluded  to. 


ANTIBACTERIAL    SERUM.  479 

In  the  original  neutral  mixture  of  crude  toxin  and  antitoxin,  both  toxin  and 
toxoid  were  present.  Any  fresh  crude  toxin  added  contains  both  toxin  and 
toxoid.  Some  of  the  fresh  toxin  turns  out  some  of  the  toxoid,  and  thus  being 
put  out  of  action  there  is  not  enough  poisonous  material  to  cause  death  if 
only  one  M.L.D.  has  been  added  to  the  neutral  mixture.  What  remains 
when  the  rearrangement  of  molecules  has  taken  place  is  not  toxin  plus 
toxoid,  but  toxoid  plus  toxoid. 

Antibacterial  Serum.  —  The  stages  in  the  preparation  of  anti- 
bacterial sera  correspond  to  those  in  the  case  of  antitoxic  sera, 
but  living,  or,  in  the  early  stages,  dead  cultures,  are  used  instead 
of  toxin  separated  by  nitration,  and  in  order  to  obtain  a  serum  of 
high  antibacterial  power  a  very  virulent  culture  in  large  doses 
must  be  ultimately  tolerated  by  the  animal.  For  this  purpose  a 
fairly  virulent  culture  is  obtained  fresh  from  a  case  of  the  par- 
ticular disease,  and  its  virulence  may  be  further  increased  by 
the  method  of  passage.  This  method  of  obtaining  a  high  degree 
of  immunity  against  the  microbe  is  specially  applicable  in  the 
case  of  those  organisms  which  invade  the  tissues  and  multiply  to 
a  great  extent  within  the  body,  and  of  which  the  toxic  effects, 
though  always  existent,  are  proportionately  small  in  relation  to 
the  number  of  organisms  present.  The  method  has  been  applied 
in  the  case  of  the  typhoid  and  cholera  organisms,  the  bacillus  of 
bubonic  plague,  the  bacillus  coli  communis,  the  pneumococcus, 
streptococcus  (Marmorek),  and  many  others.  In  fact,  it  seems 
capable  of  very  general  application. 

The  important  result  obtained  by  such  experiments  is,  that  if 
an  animal  be  highly  immunised  by  the  method  mentioned,  the 
development  of  the  immunity  is  accompanied  by  the  appearance 
in  the  blood  of  protective  substances,  which  can  be  transferred  to 
another  animal.  The  law  enunciated  by  Behring  regarding 
immunity  against  toxins  thus  holds  good  in  the  case  of  the  liv- 
ing organisms,  as  was  first  shown  by  Pfeiffer.  The  latter  found, 
for  example,  that  in  the  case  of  the  cholera  organism,  so  high  a 
degree  of  immunity  could  be  produced  in  the  guinea-pig,  that 
.002  c.c.  of  its  serum  would  protect  another  guinea-pig  against 
ten  times  the  lethal  dose  of  the  organisms,  when  injected  along 
with  them.  Here  again  is  presented  the  remarkable  potency  of 
the  antagonising  substances  in  the  serum,  which  in  this  case 
lead  to  the  destruction  of  the  corresponding  microbe. 

The  antistreptococcic  serum   of    Marmorek   may  be   briefly 


480  IMMUNITY. 

described,  as  it  has  come  into  extensive  practical  use.  This 
observer  found  that  he  could  intensify  the  virulence  of  a  strepto- 
coccus by  growing  it  alternately  in  the  peritoneal  cavity  of 
a  guinea-pig  and  in  a  mixture  of  human  blood  serum  and 
bouillon  (vide  p.  46).  The  virulence  became  so  enormously 
increased  by  this  method,  that  when  only  one  or  two  organisms 
were  introduced  into  the  tissues  of  a  rabbit  a  rapidly  fatal 
septicaemia  was  produced.  Streptococci  of  this  high  degree  of 
virulence  were  used  first  by  subcutaneous,  afterwards  by  in- 
travenous, injection,  to  develop  a  high  degree  of  resistance  in 
the  horse.  Injections  were  continued  over  a  considerable  period 
of  time,  and  the  protective  power  of  the  serum  was  tested 
by  mixing  it  with  a  certain  dose  of  the  virulent  organisms,  and 
then  injecting  into  a  rabbit.  The  serum  of  a  horse  highly 
immunised  in  this  way  constitutes  the  antistreptococcic  serum 
which  has  been  extensively  used  with  success  in  many  cases  of 
streptococcic  invasion  in  the  human  subject.1  Marmorek,  how- 
ever, found  that  this  serum  had  little  antitoxic  power,  that  is, 
could  only  protect  from  a  comparatively  small  dose  of  toxin 
obtained  by  filtration  of  cultures. 

Anti-typhoid,  anti-cholera,2  anti-pneumococcic,  anti-plague, 
and  other  sera  are  all  prepared  in  an  analogous  manner. 

Properties  of  Antibacterial  Serum.  — Within  recent  years 
it  has  been  shown  that  an  antibacterial  serum,  in  addition  to 
being  protective,  may  sometimes  also  present  important  objective 
reactions  against  the  corresponding  organism,  and  these  are  of 
high  importance,  as  they  afford  valuable  aid  in  the  study  of  the 
nature  of  the  preventive  power.  Of  such  actions  the  two  chief 
are  the  lysogenic  and  the  agglutinative. 

Lysogenic  Action.  —  Pf eiffer  found  that  if  certain  organisms, 
e.g.  the  cholera  spirillum,  were  injected  into  the  peritoneal  cavity 
of  a  guinea-pig  highly  immunised  against  these  organisms  they 
lost  their  motility  almost  immediately,  gradually  became  granular 
and  swollen  up  in  places  into  droplets,  and  then  disappeared  in 
the  fluid,  all  these  changes  sometimes  occurring  within  half  an 
hour — lysogenic  action.  Further,  he  found  that  the  same 

1  Results  in  general  are  now  considered  not  to  be  as  satisfactory  as  was  at  first 
supposed  from  the  earlier  reports  of  the  use  of  this  serum. 

2  A  true  antitoxic  cholera  serum  has  been  prepared  by  Metchnikoff,  E.  Roux, 
and  Taurelli-Salimbeni. 


PROPERTIES    OF   ANTIBACTERIAL    SERUM.  481 

phenomenon  was  witnessed  if  a  minute  quantity  of  the  anti-serum 
was  added  to  a  certain  quantity  of  the  organisms,  and  the 
mixture  injected  into  the  peritoneal  cavity  of  another  animal. 
In  both  cases  the  organisms  die  an  extracellular  death,  and  their 
destruction  is  brought  about  by  the  medium  of  a  specific  sub- 
stance in  the  anti-serum.  Pfeiffer  found  that  the  serum  of  con- 
valescent cholera  patients  gave  the  same  reaction  as  that  of 
immunised  animals.  He  obtained  the  same  reaction  also  in  the 
case  of  the  typhoid  bacillus  and  other  organisms.  From  his 
observations  he  concluded  that  the  reaction  was  specific,  and 
could  be  used  as  a  means  of  distinguishing  organisms  which 
resemble  one  another.  He  also  found  that  an  anti-serum  heated 
to  70°  C.  for  an  hour  produced  the  reaction  when  injected  with 
the  corresponding  organisms  into  the  peritoneum  of  a  fresh 
animal.  He  considered  that  the  specific  substance  in  the  serum 
existed  chiefly  in  an  inert  and  somewhat  stable  form,  and  that 
it  became  actively  bactericidal  by  the  aid  of  living  cells,  probably 
those  of  the  peritoneal  endothelium.  Metchnikoff,  however, 
showed  that  lysogenesis  occurred  when  the  bacteria  were  simply 
placed  in  some  fresh  peritoneal  fluid  to  which  the  anti-serum 
had  been  added  outside  the  body,  and  Bordet  showed  that  the 
serum  of  a  fresh  animal  could  be  substituted  for  peritoneal  fluid 
with  the  same  result.  The  latter  observer  also  found  that  in 
some  cases  the  anti-serum  alone,  if  used  quite  fresh,  could  pro- 
duce in  vitro  the  destruction  of  the  bacteria.  In  these  cases, 
accordingly,  the  action  of  the  endothelial  cells  was  excluded. 
Bordet  found  that  in  every  case  in  which  Pfeiffer's  reaction  took 
place  within  the  body  of  an  animal,  a  similar  lysogenic  reaction 
could  be  observed  by  his  method  outside  the  body. 

His  method  was  the  following:  (a}  An  emulsion  of  the  living  organisms 
(for  example,  of  the  cholera  vibrio)  was  made  by  adding  a  young  culture  to 
about  5  c.c.  of  bouillon  ;  (£)  two  drops  of  this  emulsion  were  taken,  and  mixed 
with  a  small  drop  of  anti-cholera  serum  ;  (c)  a  drop  of  this  mixture  was  taken, 
and  there  was  added  to  it  a  drop  of  equal  size  of  fresh  serum  from  a  normal 
guinea-pig.  A  hanging-drop  preparation  was  made,  and  a  change  similar  to 
that  described  by  Pfeiffer  was  observed  within  one  to  two  hours  if  the  prepara- 
tion was  kept  at  the  temperature  of  the  body. 

The  outcome  of  the  research  with  regard  to  lysogenic  action 
may  be  said  to  be  the  following.  In  order  to  produce  the  occur- 
rence of  the  phenomena,  two  substances  are  necessary.  One  is 

21 


482  IMMUNITY. 

specially  developed  during  the  process  of  immunisation,  and  gives 
the  anti-serum  its  special  character ;  it  is  usually  known  as  the 
immune-body  (Ehrlich)  or  substance  sensibilisatrice  (Bordet). 
It  is  comparatively  resistant  to  heat,  and  can  usually  be  subjected 
to  65°  C.  for  an  hour  without  being  destroyed.  It  is  apparently 
the  protective  substance,  as  an  anti-serum  does  not  lose  its  pro- 
tective power  when  heated  to  the  temperature  mentioned.  It, 
however,  cannot  produce  bacteriolysis  alone,  but  requires  for  this 
another  substance  present  in  normal  serum.  This  latter  is  more 
labile,  being  readily  destroyed  at  65°  C.,  and  even  by  half  an 
hour  at  55°  C.  ;  it  is  known  by  various  names  —  addiment  or 
complement  (Ehrlich),  alexine  or  cytase  (French  writers).  We 
shall  speak  of  the  two  substances  just  described  as  "immune- 
body"  and  ''complement"  respectively.  The  laws  of  lysogenesis 
are,  however,  not  peculiar  to  the  case  of  solution  of  bacteria  by 
the  fluids  of  the  body,  but,  as  has  been  shown  within  the  last 
few  years,  hold  also  in  the  case  of  other  organised  substances, 
red  corpuscles,  leucocytes,  etc.,  when  these  are  introduced  into 
the  tissues  of  an  animal  as  in  a  process  of  immunisation.  Of 
such  sera  the  haemolytic  have  been  most  fully  studied,  and  have 
been  the  means  of  throwing  much  light  on  the  process  of  lyso- 
genesis, and  thus  on  one  part  of  the  subject  of  immunity.  A 
short  account  of  their  properties  may  now  be  given. 

Hcemolytic  and  other  Sera.  —  It  has  been  known  for  some 
time  that  in  some  instances  the  blood  serum  of  one  animal  has, 
in  certain  degree,  the  power  of  dissolving  the  red  corpuscles  of 
another  animal  of  different  species ;  in  other  instances,  how- 
ever, this  property  cannot  be  detected.  Bordet  showed  that 
if  one  animal  were  treated  with  repeated  injections  of  the 
corpuscles  of  another,  the  serum  of  the  former  acquired  a 
marked  haemolytic  property  towards  the  corpuscles  of  the  latter, 
the  property  being  demonstrated  when  the  serum  is  added  to 
the  corpuscles.  A  mixture  of  five  parts  of  defibrinated  blood, 
which  of  course  contains  the  corpuscles,  and  ninety-five  parts 
of  .75  per  cent  chloride  of  sodium  solution  is  used,  and  to 
this  varying  quantities  of  the  haemolytic  serum  are  added  and 
allowed  to  stand  for  some  time  at  a  warm  temperature,  usually 
for  one  hour  at  37°  C.  Bordet  also  found  that  the  haemolytic 
property  disappeared  when  the  haemolytic  serum  was  heated 
at  55°  C.,  but  was  regained  on  the  subsequent  addition  of 


H^MOLYTIC   AND   OTHER   SERA.  483 

some  serum  from  a  fresh  (i.e.  non-treated)  animal.  These 
observations  have  been  fully  confirmed,  and  it  may  be  stated 
that  in  each  case  the  haemolytic  property  is  "practically 
specific,"  i.e.  is  exerted  only  towards  the  corpuscles  used  in  the 
injections ;  moreover,  by  the  injection  of  corpuscles  from  more 
than  one  species  of  animal,  a  serum  with  multiple  haemolytic 
properties  may  be  obtained.  Ehrlich  and  Morgenroth  analysed 
the  phenomena  in  question,  and  showed  that  the  specially 
developed  and  heat-resisting  substance,  "immune-body,"  entered 
into  combination  with  the  red  corpuscles  at  a  comparatively  low 
temperature.  This  was  shown  by  adding  the  heated  serum  to 
the  red  corpuscles  in  salt  solution  (of  course  no  haemolysis 
occurs),  and  after  some  time  centrifugalising  the  mixture.  On 
separating  the  corpuscles  it  was  found  that  the  haemoglobin  was 
set  free  on  the  addition  of  some  serum  from  a  fresh  animal ;  it 
was  also  found  that  the  immune-body  was  absent  from  the  clear 
fluid.  In  other  words,  the  red  corpuscles  fix  or  become  com- 
bined with  the  immune-body.  In  a  corresponding  manner  they 
came  to  the  conclusion  that  the  immune-body  combined  with  the 
complement  (in  normal  serum),  though  the  combination  was  less 
firm  and  only  occurred  at  a  higher  temperature  —  best  about 
37°  C.  They  therefore  consider  that  the  immune-body  acts  as  a 
sort  of  connecting  link  between  the  red  corpuscle  and  the  com- 
plement. Bordet,  on  the  other  hand,  holds  that  the  immune- 
body  acts  merely  as  a  sensitising  agent  —  hence  the  term  sub- 
stance sensibilisatrice  —  and  allows  the  ferment-like  complement 
to  act.  Regarding  the  important  fact  that  in  the  case  of  each 
anti-serum  of  this  group  a  special  immune-body  is  developed, 
and  that  the  laws  of  haemolysis  are  identical  with  those  of 
bacteriolysis,  practically  all  are  agreed.  It  is  a  disputed  point 
whether  there  are  several  distinct  complements  in  a  normal 
serum  with  different  relations  to  different  immune-bodies,  for 
which  Ehrlich  and  his  co-workers  have  brought  forward  a  large 
amount  of  evidence,  or  whether,  as  Bordet  holds,  there  is  a 
single  complement,  which  may,  however,  show  slight  variations 
in  behaviour  towards  different  immune  bodies.  Workers  of  the 
French  school  also  hold  that  complement  does  not  exist  in  the 
free  condition  in  the  blood,  but  is  liberated  from  the  leucocytes 
when  the  blood  is  shed ;  though  this  cannot  be  held  as  proved, 
there  is  evidence  that  the  amount  of  free  complement  in- 


484  IMMUNITY. 

creases  after  the  blood  is  shed  and  some  time  later  gradually 
diminishes. 

In  addition  to  haemolytic  sera,  anti-sera  have  been  obtained 
by  the  injection  of  leucocytes,  spermatozoa,  ciliated  epithelium, 
liver  cells,  nervous  tissue,  etc.  The  laws  governing  the  pro- 
duction and  properties  of  these  are  identical,  that  is,  each  serum 
exhibits  a  specific  property  towards  the  body  used  in  its  produc- 
tion —  i.e.  dissolves  leucocytes,  immobilises  spermatozoa,  etc. 
It  may  also  be  mentioned  that  each  anti-serum  usually  exhibits 
toxic  properties  towards  the  animal  whose  cells  have  been  used 
in  the  injections,  e.g.  a  haemolytic  serum  may  produce  a  fatal 
result,  with  signs  of  extensive  blood  destruction,  haemoglobinuria, 
etc.,  i.e.  it  is  haemotoxic  for  the  particular  animal ;  a  serum 
prepared  by  injection  of  liver  cells  has  been  found  to  produce  on 
injection  necrotic  changes  in  the  liver  in  the  species  of  animal 
whose  liver  cells  were  used.  These  are  mentioned  as  examples 
of  a  very  large  group  of  specific  activities. 

With  regard  to  the  sites  of  origin  of  immune-bodies  our 
information  is  still  very  deficient,  Pfeiffer  and  Marx  brought 
forward  evidence  in  the  case  of  typhoid,  and  Wassermann  in  the 
case  of  cholera,  that  the  immune-bodies  are  chiefly  formed 
in  the  spleen,  lymphatic  glands,  and  bone-marrow.  According 
to  certain  workers  of  the  French  school,  the  chief  source  of 
sera  acting  on  cells  such  as  red  blood  corpuscles  is  the  large 
mononucleated  leucocytes,  whilst  sera  acting  on  bacteria  are 
chiefly  derived  from  the  polymorpho-nuclear  leucocytes.  The 
active  bodies  in  the  former  are  by  these  observers  sometimes 
spoken  of  as  " macrocytases"  those  of  the  latter  as  " microcy- 
tases"  Another  view  is  that  immune-bodies  are  chiefly  formed 
by  the  large  mononucleated  leucocytes,  whilst  complements  are 
products  of  the  polymorpho-nuclears.  That  these  cells  are  con- 
cerned in  the  production  of  antagonistic  and  protective  sub- 
stances is  almost  certain,  though  another  possible  source  of  wide 
extent,  viz.,  the  endothelium  of  the  vascular  system,  has  been 
largely  overlooked.  As  yet,  definite  statements  cannot  be  made 
on  this  point. 

Agglutination.  —  Charrin  and  Roger  in  1889  observed  that 
when  the  bacillus  pyocyaneus  was  grown  in  the  serum  of  an 
animal  immunised  against  this  organism,  the  growth  formed  a 
deposit  at  the  foot  of  the  vessel ;  whereas  a  growth  in  normal 


AGGLUTINATION.  485 

serum  produced  a  uniform  turbidity.  Gruber  and  Durham,  in 
investigating  Pfeiffer's  reaction,,  discovered  an  analogous  phe- 
nomenon. They  found  that  when  a  small  quantity  of  the  serum 
of  an  animal  highly  immunised  against  a  particular  motile 
organism  (cholera  vibrio,  typhoid  bacillus,  etc.)  is  added  to  an 
emulsion  of  the  organisms,  the  latter  lose  their  motility  and 
become  agglutinated  into  clumps.  In  a  small  test-tube  a  reac- 
tion in  this  way  occurs  which  is  visible  to  the  naked  eye,  a  sort 
of  precipitate  forming  which  consists  of  masses  of  the  bacteria. 
Non-motile  organisms  also  may  be  agglutinated  by  the  corre- 
sponding serum,  as  may  also  red  corpuscles  by  a  hsemolytic 
serum.  As  a  rule,  the  higher  the  degree  of  immunity  the  smaller 
is  the  amount  of  serum  necessary  to  produce  agglutination. 
The  phenomenon  depends  upon  the  presence  of  definite  bodies 
in  the  serum  called  agglutinins.  In  each  case  these  can  only 
clump  a  certain  amount  of  bacteria,  and  are  used  up  in  the 
process,  apparently  by  a  combination  with  the  bacteria,  probably 
attended  with  a  physical  change  in  the  envelopes  of  the  latter, 
and  this  Gruber  and  Durham  consider  forms  the  essential  part 
of  Pfeiffer's  reaction. 

The  observations  just  described  have  led  to  the  discovery 
of  the  method  of  serum  diagnosis  of  disease,  which  has  been 
applied  especially  to  typhoid  fever,  as  already  detailed  (vide 
p.  340).  It  had  been  already  found  that  the  serum  of  conva- 
lescents from  typhoid  fever  could  protect  animals  to  a  certain 
extent  against  typhoid  fever,  and,  in  view  of  the  facts  experi- 
mentally established,  it  appeared  a  natural  proceeding  to  inquire 
whether  such  serum  possessed  an  agglutinative  action  and  at 
what  stage  of  the  disease  it  appeared.  The  result,  obtained 
independently  by  Grunbaum  and  Widal,  but  first  published  by 
the  latter,  was  to  show  that  the  serum  possessed  this  specific 
action  long  before  the  cure  of  the  disease,  in  fact  shortly  after 
infection  had  taken  place.  It  is  probable  that  it  depends  upon 
a  process  of  immunisation  developing  from  an  early  stage  of  the 
disease.  Agglutination  is  also  observed  in  the  case  of  cholera, 
Malta  fever,  bacillary  dysentery,  glanders,  plague,  infection  by 
Gaertner's  bacillus,  B.  coli,  etc. 

The  physical  changes  on  which  agglutination  depends  cannot 
as  yet  be  said  to  be  fully  understood.  As  stated  above,  Gruber 
and  Durham  considered  that  the  agglutinin  produced  a  change 


486  IMMUNITY. 

in  the  envelope  of  the  .bacterium,  causing  it  to  swell  up  and 
become  viscous,  and  there  are  certain  facts  in  favour  of  this  view. 
On  the  other  hand,  this  is  probably  not  the  full  explanation,  as  it 
has  been  shown  by  Nicolle  and  by  Kruse  that  if  an  old  bacterial 
culture  be  filtered  through  porcelain,  the  addition  of  some  of 
the  corresponding  serum  produces  some  change  in  it,  so  that 
even  minute  inorganic  particles  become  aggregated  into  clumps. 
The  phenomenon  would  thus  appear  to  be  the  result  of  the 
interaction  of  the  agglutinin  and  some  substance  in  the  bacterial 
cell  (which  substance  evidently  leads  to  the  development  of  the 
agglutinin  in  the  living  body) ;  and,  as  Duclaux  states,  is  closely 
allied  to  a  process  of  coagulation.  Of  greater  importance,  how- 
ever, is  the  relationship  of  agglutination  to  immunity.  Gruber 
and  Durham  considered  that  agglutination  was  the  essential 
part  of  Pfeiffer's  reaction  or  lysogenesis,  the  change  produced  in 
the  bacteria  allowing  the  bactericidal  action  naturally  possessed 
by  the  serum  to  come  into  play.  Others,  again,  consider  that 
the  two  are  independent  of  one  another.  The  fact  that  the 
agglutinative  power  appears  early  in  an  infective  disease  is  often 
pointed  to  as  proof  of  such  a  view.  This  line  of  reasoning  is 
not,  however,  by  itself  conclusive,  as  we  must  suppose  that  the 
reaction,  or  series  of  reactions,  leading  to  immunity  begins  at  an 
early  period  and  gradually  increases  until  cure  results.  It  is 
also  to  be  noted  that  agglutinins  accord  with  protective  sub- 
stances as  regards  resistance  to  heat  (i.e.  a  serum  heated  to  55°  C. 
loses  its  bactericidal  or  lysogenic  power  while  the  agglutinative 
and  protective  properties  remain,  vide  p.  482).  On  the  other 
hand,  a  serum  may  be  highly  protective  without  being  agglutina- 
tive, and  when  the  two  properties  are  present  together  they  do 
not  always  run  in  the  same  proportion.  It  is  doubtful,  however, 
whether  a  serum  ever  possesses  a  high  degree  of  agglutination 
without  having  some  protective  power.  On  the  whole  it  seems 
safe  to  say  that  agglutinins  and  immune-bodies,  though  not 
necessarily  identical,  are  the  products  of  corresponding  reactive 
processes,  and  their  formation  is  governed  by  corresponding  laws. 
The  bacterial  cell,  containing  as  it  does  various  complicated 
organic  constituents,  may  cause  the  formation  of  more  than  one 
anti-substance,  and  each  of  these  has  a  combining  affinity  for 
part  of  the  bacterial  body.  Agglutination  is  most  probably  to 
be  regarded  as  a  phenomenon  prejudicial  to  the  corresponding 


VARIOUS   ACTIONS   OF    SPECIFIC    SERA.  487 

bacterium,  and  thus  in  nature  to  be  allied  with  the  process  of 
immunisation. 

Besides  those  stated  above,  other  phenomena  have  been  ob- 
served in  the  interaction  of  anti-sera  and  the  corresponding 
bacteria.  For  example,  it  has  been  shown  that  when  certain 
bacteria  —  e.g.  the  typhoid  bacillus,  B.  coli,  and  B.  proteus  — 
are  grown  in  bouillon  containing  a  small  proportion  of  the 
homologous  serum,  their  morphological  characters  may  be 
altered,  growth  taking  place  in  the  form  of  threads  or  chains 
which  are  not  observed  in  ordinary  conditions.  In  other  in- 
stances a  serum  may  inhibit  some  of  the  vital  functions  of  the 
corresponding  bacterium. 

Summary  with  regard  to  Anti-sera.  —  In  a  former  chapter  it 
has  been  shown  that  in  the  production  of  disease  by  bacteria 
there  are  two  main  factors  concerned,  viz.,  the  multiplication  of 
the  living  organisms  in  the  tissues  and  the  production  by  them 
of  toxins.  The  facts  which  have  been  stated  above  show  that  in 
the  blood  serum  of  highly  immunised  animals  there  are  present 
substances  of  remarkable  potency  which  may  act  against  either 
of  these  two  factors.  In  the  first  place,  a  serum  may  protect 
against  the  separated  toxin,  or,  in  other  words,  may  be  antitoxic. 
In  the  second  place,  a  serum  may  lead  to  the  destruction  of 
the  organisms  ;  the  term  antibacterial  is,  therefore,  conveniently 
applied  to  such  a  serum.  In  many  instances  an  antibacterial 
serum  has  little  or  no  effect  against  the  toxins.  The  action  of 
both  varieties  of  anti-sera  is,  within  certain  limits,  specific,  being 
exerted  only  against  the  particular  organism  or  toxin  which  has 
been  used  in  its  preparation,  having  a  definite  value  which  can 
be  ascertained  by  experiment.  It  does  not  follow  from  what  has 
been  said  that  a  serum  may  not  act  in  both  of  the  ways  described. 
A  given  serum  might,  for  example,  be  powerfully  antibacterial 
and  feebly  antitoxic  at  the  same  time. 

It  is  specially  to  be  noted  that  anti-sera  are  not  peculiar  to  the 
case  of  bacteria  and  their  toxins,  but  constitute  a  large  group, 
the  characteristic  features  of  which,  in  general  terms,  are,  that 
they  are  produced  by  the  injection  of  complicated  organic 
substances,  either  in  solution  or  as  formed  elements.  This  group 
may  be  conveniently  divided  into  two  great  classes.  In  one  of 
these  the  characteristic  effect  is  apparently  due  to  one  substance 
acting  alone,  whilst  in  the  other  this  effect  requires  for  its  com- 


488  IMMUNITY. 

pletion  substances  normally  present  in  the  serum  (complements). 
Antitoxic  sera  belong  to  the  first  group,  antibacterial  sera  to  the 
second.  In  all,  however,  the  substance  specifically  developed 
appears  to  have  a  combining  affinity  for  the  substance  introduced 
into  the  body  —  toxin,  albumin,  bacterium,  animal  cell,  etc.,  as 
the  case  may  be. 

Therapeutic  Effects  of  Anti-sera.  — As  will  have  been  gathered, 
the  chief  human  diseases  treated  by  anti-sera  are  diphtheria,  te- 
tanus, streptococcus  infection,  pneumonia,  plague,  and  snake 
bite.  Of  the  results  of  such  treatment  most  is  known  in  the 
case  of  diphtheria.  Here  a  very  great  diminution  in  the  mortality 
has  resulted.  The  diphtheria  antitoxin  came  into  general  use 
about  October,  1894,  and  the  statistics  published  by  Behring 
towards  the  end  of  1895  indicate  results  which  have  since  been 
confirmed.  In  the  Berlin  Hospitals  the  average  mortality  for 
the  years  1891-93  was  36.1  per  cent,  in  1894  it  was  21.1  per 
cent,  and  in  January-July,  1895,  14.9  per  cent.  The  objection 
that  in  some  epidemics  a  very  mild  type  of  disease  prevails  is 
met  by  the  fact  that  similar  diminutions  of  mortality  have 
occurred  all  over  the  world.  Loddo  collected  the  results  of  7000 
cases  in  Europe,  America,  Australia,  and  Japan,  in  which  the 
mortality  was  20  per  cent  as  compared  with  a  former  mortality 
in  the  same  hospitals  of  44  per  cent.  It  has  also  been  observed 
that  if  during  an  epidemic  the  supply  of  serum  fails,  the  mortality 
at  once  rises  ;  and  in  two  instances  recorded  it  was  doubled.  It 
must  here  be  remembered  that  from  the  spread  of  bacteriological 
knowledge  the  diagnosis  of  diphtheria  is  now  much  more  accurate 
than  formerly.  Another  effect  of  the  antitoxic  treatment  has 
been  that  when  tracheotomy  is  necessary  the  percentage  of 
recoveries  is  now  much  higher,  being  73  per  cent  instead  of  27 
per  cent  in  a  group  of  cases  collected  by  the  American  Pediatric 
Society.  In  the  London  fever  hospitals  since  1894  the  recoveries 
after  tracheotomy  have  been  56.4  as  compared  with  32.1  per 
cent  previous  to  the  introduction  of  antitoxin.  One  of  the  most 
striking  results  obtained  in  the  same  hospitals  is  a  reduction  of 
the  death-rate  in  post-scarlatinal  diphtheria  from  50  per  cent  to 
between  4  per  cent  and  5  per  cent.  As  the  disease  here  occurs 
while  the  patient  is  under  observation  the  treatment  is  nearly 
always  begun  on  the  first  day.  It  is  a  matter  of  prime  importance 
that  the  treatment  should  be  commenced  whenever  the  disease  is 


THEORIES    AS   TO   ACQUIRED   IMMUNITY.  489 

recognised.  Behring  showed  that  in  cases  treated  on  the  first  and 
second  days  of  the  disease  the  mortality  was  only  7.3  per  cent, 
and  this  has  been  generally  confirmed,  whilst  after  the  fifth  day  it 
was  of  little  service  to  apply  the  treatment.  In  order  to  obtain 
such  results  it  cannot  be  too  strongly  insisted  on  that  attention 
should  be  given  to  the  dosage.  When  bad  results  are  obtained 
it  may  be  strongly  suspected  that  this  precaution  has  not  been 
observed.  In  the  treatment  of  acute  tetanus  by  the  antitoxin 
the  improvement  in  results  has  not  been  marked,  but  some 
chronic  cases  have  been  benefited.  In  the  case  of  Yersin's  anti- 
plague  serum,  though  benefit  has  appeared  to  follow  its  use, 
experience  with  its  effects  has  been  too  limited  to  enable  a 
judgment  to  be  formed.  The  same  may  be  said  to  be  true  of 
the  antistreptococcic  and  antipneumonic  sera,  and  also  of  anti- 
venin,  though  in  the  case  of  the  first  mentioned  numerous  cases 
of  apparently  successful  result  have  been  recorded. 

As  has  been  shown  above,  antibacterial  sera  require  for  their 
complete  action  a  sufficiency  of  complements,  and  as  these 
diminish  in  amount  when  a  serum  is  kept,  the  unsatisfactory 
results  with  this  class  of  sera  may  be  due  to  a  deficiency  of 
complement.  Or  it  may  be  as  Ehrlich  has  suggested,  that  the 
complement  naturally  existing  in  human  serum  does  not  suit  the 
immune-body  in  the  anti-serum.  There  is  no  doubt,  however, 
that  this  question  of  comple'ments  is  one  of  importance,  and  will, 
in  all  probability,  be  cleared  up  in  the  further  development  of 
research  on  this  subject. 

Theories  as  to  Acquired  Immunity. 

The  advances  made  within  recent  years  in  our  knowledge 
regarding  artificial  immunity  and  the  methods  by  which  it  may 
be  produced  have  demonstrated  the  insufficiency  of  various 
theories  which  had  been  propounded.  Only  a  short  reference 
need  be  made  to  these.  The  theory  of  exhaustion,  with  which 
Pasteur's  name  is  associated,  supposed  that  in  the  body  of  the 
living  animal  there  are  substances  necessary  for  the  existence  of 
a  particular  organism,  which  become  used  up  during  the  sojourn 
of  that  organism  in  the  tissues ;  this  pabulum  being  exhausted, 
the  organisms  die  out.  Such  a  supposition  is,  of  course,  quite 
disproved  by  the  facts  of  passive  immunity.  According  to  the 
theory  of  retention,  the  bacteria  within  the  body  were  considered 


490  IMMUNITY. 

to  produce  substances  which  are  inimical  to  their  growth,  so  that 
they  die  out,  just  as  they  do  in  a  test-tube  culture  before  the 
medium  is  really  exhausted.  Such  a  theory  only  survives  now 
in  the  view  that  antitoxins  are  modified  toxins,  the  evidence 
against  which  has  already  been  discussed  (p.  475).  There  then 
came  the  humoral  theory  and  the  tJieory  of  phagocytosis,  but 
neither  of  these  is  tenable  in  its  pure  form,  and  the  distinction 
between  them  need  not  be  maintained.  For  on  the  one  hand, 
any  substance  with  specific  property  in  the  serum  must  be  the 
product  of  cellular  activity,  and  on  the  other  hand,  the  facts 
with  regard  to  passive  immunity  go  far  beyond  the  ingestive  and 
digestive  properties  of  phagocytes,  though  these  cells  may  be  in 
part  the  source  of  important  bodies  in  the  serum.  At  the  present 
time  interest  centres  around  two  theories,  viz.,  Ehrlich's  side- 
chain  theory  and  Metchnikoff's  phagocytic  theory  as  further 
developed.  These  will  now  be  discussed,  and  it  may  be  noted 
that  the  ground  covered  by  each  is  not  coextensive.  For  the 
former  deals  chiefly  with  the  production  of  anti-substances  and 
its  biological  significance,  the  latter  deals  with  the  defensive  prop- 
erties of  cells,  either  directly  by  their  phagocytic  activity  or 
indirectly  by  substances  produced  by  them  after  the  manner  of 
digestive  ferments.  It  will  be  seen,  however,  that  each  has  a 
normal  process  as  its  basis,  viz.,  that  of  nutrition. 

i.  Ehrlich's  Side-chain  Theory.  —  This  may  be  said  to  be  an 
application  of  his  views  regarding  the  nourishment  of  protoplasm. 
A  molecule  of  protoplasm  (in  the  general  sense)  may  be  regarded 
as  composed  of  a  central  atom-group  (Leistungskern)  with  a 
large  number  of  side-chains  (Seitenketten),  i.e.  atom  groups  with 
combining  affinity  for  food-stuffs.  It  is  by  means  of  these  latter 
that  the  living  molecule  is  increased  in  the  process  of  nutrition, 
and  hence  the  name  receptors  given  by  Ehrlich  is  on  the  whole 
preferable.  These  receptors  are  of  two  chief  kinds :  the  first 
has  a  single  unsatisfied  combining  group  and  fixes  molecules  of 
simpler  constitution  —  receptor  of  the  first  order ;  the  second  has 
two  such  groups,  one  for  the  food  molecule  and  another  which 
fixes  a  ferment  in  the  fluid  medium  around  —  receptor  of  the 
second  order  or  amboceptor.  These  latter  receptors  come  into 
action  in  the  case  of  larger  food  molecules  which  require  to 
be  broken  up  by  ferment  action  for  the  purposes  of  the  cell 
economy.  In  considering  the  application  of  this  idea  to  the 


EHRLICH'S    SIDE-CHAIN    THEORY. 


491 


facts  of  passive  immunity,  it  must  be  kept  in  view  that  all  the 
substances  for  which  anti-substances  have  been  obtained  are,  like 
proteids,  of  unknown  but  undoubtedly  of  very  complex  chemical 
constitution,  and  that  in  apparently  every  case  the  anti-substance 
enters  into  combination  with  its  corresponding  substance.  The 
dual  constitution  of  toxins  and  kindred  substances,  as  already 
described  (p.  4/7),  is  also  of  importance  in  this  connection. 
Now  when  toxins  are  introduced  into  the  system  they  are  fixed, 
like  food-stuffs,  by  their  haptophorous  groups  to  the  receptors  of 
the  cell  protoplasm.  If  they  are  in  sufficiently  large  amount  the 
toxophorous  part  of  the  toxin  molecule  produces  that  disturbance 
of  the  protoplasm  which  is  shown  by  symptoms  of  poisoning. 
If,  however,  they  are  in  smaller  dose,  as  in  the  early  stages  of 
immunisation,  fixation  to  the  protoplasm  occurs  in  the  same  way  ; 
and  as  the  combination  of  receptors  with  toxin  is  supposed  to 
be  of  firm  nature,  the  receptors  are  lost  for  the  purposes  of  the 
cell,  and  the  combination  R.-T.  (receptor  +  toxin)  is  shed  off  into 
the  blood.  The  receptors  thus  lost  become  replaced  by  new 
ones,  and  when  additional  toxin  molecules  are  introduced,  these 
new  receptors  are  used  up  in  the  same  manner  as  before.  As 
a  result  of  this  repeated  loss  the  regeneration  of  the  receptors 
becomes  an  over-regeneration,  and  the  receptors  formed  in  excess 
appear  in  the  free  condition  in  the  blood  stream  and  then  consti- 
tute antitoxin  molecules.  So  that  these  receptors  which,  when 
forming  part  of  the  cell  protoplasm,  anchor  the  toxin  to  the  cell, 
and  thus  are  essential  to  the  occurrence  of  toxic  phenomena,  in 
the  free  condition  unite  with  the  toxin  and  thus  the  toxin  can 
no  longer  combine  with  the  cells  and  exert  a  pathogenic  action. 
Antitoxin  mole  cities  are  thus  free  receptors  of  tJie  first  order.  A 
corresponding  explanation  applies  to  the  origin  of  antibacterial 
and  like  sera.  The  molecules  of  bacterial  bodies,  of  stromata  of 
red  corpuscles,  etc.,  act  as  unsuitable  food-stuffs  to  the  cells  and 
use  up  the  receptors  which  combine  with  them.  These  molecules 
are  chemically  of  larger  size  than  the  toxin  molecules,  and  the 
corresponding  receptors  are  those  which  can  also  fix  a  ferment. 
The  immune  bodies  of  antibacterial,  Jicemolytic,  and  other  like 
sera  are  thus  free  receptors  of  the  second  order.  Ehrlich  does  not 
state  what  cells  are  specially  concerned  in  the  production  of 
anti-substances,  but  from  what  has  been  stated  it  is  manifest  that 
any  cell  which  fixes  a  toxin  molecule,  for  example,  is  potentially 


492  IMMUNITY. 

a  source  of  antitoxin.  Cells,  to  whose  disturbance,  resulting- 
from  the  fixation  of  toxin,  characteristic  symptoms  of  poisoning 
are  due,  will  thus  be  sources  of  antitoxin,  e.g.  cells  of  the  nervous 
system  in  the  case  of  tetanus,  though  the  cells  not  so  seriously 
affected  by  toxin  fixation  may  act  in  the  same  way. 

When  we  come  to  consider  how  far  Ehrlich's  theory  is  in 
harmony  with  known  facts,  we  find  that  there  is  much  in  its 
favour.  In  the  first  place,  it  explains  the  difference  between 
active  and  passive  immunity,  e.g.  difference  in  duration,  etc.  ;  in 
the  former  the  cells  have  acquired  the  habit  of  discharging  anti- 
substances,  in  the  latter  the  anti-substances  are  simply  present 
as  the  result  of  direct  transference.  It  is  also  in  harmony  with 
the  action  of  antitoxins,  etc.,  as  detailed  above,  and  especially  it 
affords  an  explanation  of  the  multiplicity  of  anti-substances. 
For,  if  we  take  the  case  of  antitoxins,  we  see  that  this  depends 
upon  the  combining  affinity  of  the  toxin  for  certain  of  the  cells 
of  the  body,  and  this  again  is  referred  back  to  the  complicated 
constitution  of  living  protoplasm.  Furthermore,  the  biological 
principle  involved  is  no  new  one,  being  simply  that  of  over- 
regeneration  after  loss. 

It  is  to  be  noted,  however,  that  it  does  not  explain  active 
immunity  apart  from  the  presence  of  anti-substances  in  the 
serum.  For  example,  an  animal  may  be  able  to  withstand  a 
much  larger  amount  of  toxin  than  could  be  neutralised  by  the 
total  amount  of  antitoxin  in  its  serum.  It  is  difficult  to  see 
what  condition  of  the  receptors  of  the  cells  would  explain  such 
a  fact,  and  the  question  arises  whether  there  may  not  be  really 
an  increased  resistance  of  the  cells  to  the  toxophorous  affinities. 
Further,  when  the  serum  of  an  animal  contains  a  large  amount 
of  antitoxin,  how  does  the  toxin  reach  the  cells  in  order  to  influ- 
ence them  as  we  know  it  does  ?  This  is  difficult  to  understand 
unless  the  toxin  has  a  greater  affinity  for  the  receptors  in  the 
cells  than  for  the  free  receptors  (antitoxin)  in  the  serum.  A 
supersensitiveness  of  the  nerve  cells  of  an  animal  to  tetanus 
toxin,  sometimes  observed  even  when  there  is  a  large  amount  of 
antitoxin  in  the  serum,  has  been  often  brought  forward  as  an 
objection.  But  this  also  may  perhaps  be  explained  by  there 
having  occurred  a  partial  damage  of  the  cell  protoplasm  by  the 
toxophorous  action  in  the  process  of  immunisation  —  an  expla- 
nation which,  of  course,  demands  that  in  some  way  the  freshly 


THE   THEORY   OF   PHAGOCYTOSIS.  493 

introduced  toxin  may  reach  the  cells  in  spite  of  the  antitoxin  in 
the  blood.  Further  investigation  alone  will  settle  these  and 
various  other  disputed  points.  At  present  we  may  say,  however, 
that  Ehrlich's  theory  is  the  only  one  which  even  attempts  to 
explain  the  cardinal  facts  of  this  aspect  of  immunity. 

2.  The  Theory  of  Phagocytosis.  —  This  theory,  brought  for- 
ward by  Metchnikoff  to  explain  the  facts  of  natural  and  acquired 
immunity,  has  been  of  enormous  influence  in  stimulating  re- 
search on  the  subject.  Looking  at  the  subject  from  the  stand- 
point of  the  comparative  anatomist,  he  saw  that  it  was  a  very 
general  property  possessed  by  certain  cells  throughout  the 
animal  kingdom,  that  they  should  take  up  foreign  bodies  into 
their  interior  and  in  many  cases  digest  and  destroy  them.  On 
extending  his  observations  to  what  occurred  in  disease,  he  came 
to  the  conclusion  that  the  successful  resistance  of  an  animal 
against  bacteria  depended  on  the  activity  of  certain  cells  called 
phagocytes.  In  the  human  subject  he  distinguished  two  chief 
varieties,  namely  (a)  the  microphages,  which  are  the  "  poly- 
morpho-nuclear,"  finely  granular  leucocytes  of  the  blood,  and  (b) 
the  macrophages,  which  include  the  larger  hyaline  leucocytes, 
endothelial  cells,  connective  tissue  corpuscles,  and,  in  short,  any 
of  the  larger  cells  which  have  the  power  of  ingesting  bacteria. 
Insusceptibility  to  a  given  disease  is  indicated  by  a  rapid  activity 
on  the  part  of  the  phagocytes,  different  varieties  being  con- 
cerned in  cfirferent  cases,  ^ —  an  activity  which  may  rapidly  de- 
stroy the  bacteria  and  prevent  even  local  damage.  If  the 
organisms  are  introduced  into  the  tissues  of  a  moderately  sus- 
ceptible animal,  there  occurs  an  inflammatory  reaction  with  local 
leucocytosis,  which  results  in  the  intracellular  destruction  of  the 
invading  organisms. '  Phagocytosis  is  regarded  by  Metchnikoff 
as  the  essence  of  inflammation.  He  also  showed  that  the 
bacteria  may  be  in  a  living  and  active  state  when  they  are 
ingested  by  leucocytes.  On  the  other  hand,  he  found  that  in 
a  susceptible  animal  phagocytosis  did  not  occur  or  was  only 
imperfect.  He  also  showed  that  when  a  naturally  susceptible 
animal  was  immunised,  the  process  was  accompanied  by  the 
appearance  of  an  active  phagocytosis.  The  ingestion  of  bac- 
teria by  phagocytes  is  undoubtedly  a  phenomenon  of  the  great- 
est importance  in  the  defence  of  the  organism.  It  is  known  that 
amoebae  and  allied  organisms  have  digestive  properties  which 


494  IMMUNITY. 

are  specially  active  towards  bacteria,  and  from  what  can  be 
directly  observed,  as  well  as  indirectly  inferred,  there  can  be  no 
doubt  that  such  a  faculty  is  also  possessed  by  the  phagocytes 
of  the  body.  Thus  bacteria  within  these  cells  are  in  a  position 
favourable  to  their  destruction.  It  is  manifest  that  chemiotaxis, 
which  regulates  the  ingestion  of  bacteria,  is  a  highly  impor- 
tant factor.  *An  animal  whose  leucocytes  are  attracted  by 
the  bacteria  will  be  in  a  more  favourable  position  than  one 
in  which  this  attraction  does  not  obtain.  In  the  process  of 
immunisation  of  a  susceptible  animal  we  see  a  negative  or 
neutral  chemiotaxis  becoming  replaced  by  positive  chemiotaxis. 
This  is  explained  by  Metchnikoff  as  due  to  an  education  or 
stimulation  of  the  phagocytes.  It  is,  however,  difficult  to  see 
how  they  can  be  stimulated  to  move  in  a  particular  direction, 
viz.,  towards  the  bacteria,  and  it  seems  more  likely  that  in  the 
fluids  of  the  immune  animal  the  bacteria  undergo  some  change 
by  which  they  can  exert  a  positive  chemiotaxis.  This  is  ren- 
dered the  more  likely  by  an  experiment  by  Denys,  in  which 
he  showed  that  in  a  hanging-drop  preparation  the  rabbit's 
leucocytes  behave  indifferently  towards  pneumococci,  whereas 
on  the  addition  of  some  antipneumococcic  serum  they  moved 
towards  the  pneumococci  and  ingested  them.  That  the  addition 
of  the  corresponding  immune  body  can  change  the  chemiotactic 
phenomena  in  this  way  can  be  readily  shown  in  the  case  of  red 
corpuscles. 

The  digestive  ferments  of  phagocytes  or  cytases  are,  according 
to  Metchnikoff,  retained  within  the  cells  under  normal  condi- 
tions, but  are  set  free  when  these  cells  are  injured,  for  example, 
when  the  blood  is  shed.  They  then  become  free  in  the  serum 
by  the  breaking  up  of  the  cells  —  the  process  known  as  pha- 
golysis  —  and  they  then  constitute  the  alexines,  or  complements 
of  Ehrlich.  Of  these,  as  has  already  been  said,  he  thinks  there- 
are  probably  two  kinds  —  one  called  macrocytase,  contained  in 
the  macrophages,  which  is  specially  active  towards  the  formed 
elements  of  the  animal  body,  protozoa,  etc. ;  and  the  other, 
microcytase,  contained  within  the  polymorphic-nuclear  leucocytes, 
which  has  a  special  digestive  action  on  bacteria.  It  is  the  micro- 
cytase  which  gives  blood  serum  its  bactericidal  properties. 

When  the  properties  of  antibacterial  sera,  as  above  described, 
are  considered  in  relation  to  phagocytosis,  Metchnikoff  gives 


THE   THEORY    OF   PHAGOCYTOSIS. 


495 


the  following  explanation.  He  admits  that  the  immune-body 
is  fixed  by  the  bacteria  (or  red  corpuscles,  as  the  case  may  be), 
though  he  does  not  state  that  a  chemical  combination  takes 
place;  hence  he  calls  it  a  fixative  (fixateur).  The  immune- 
bodies  are  to  be  regarded  as  auxiliary  ferments  {ferments  adju- 
vants} which  aid  the  action  of  the  alexine.  Unlike  the  latter, 
however,  they  are  formed  in  excess  during  immunisation  and 
set  free  in  the  serum.  He  compares  their  action  to  that  of  en- 
terokynase,  a  ferment  which  is  produced  in  the  intestine  and 
aids  the  action  of  trypsin.  Thus,  when  the  bacteria  have  fixed 
the  immune-body  their  digestion  is  facilitated  either  within  the 
phagocytes,  or  outside  of  them  when  the  alexine  has  been  set 
free  by  phagolysis.  He,  however,  maintains  that  extracellular 
digestion  or  lysogenesis  does  not  take  place  without  the  occur- 
rence of  phagolysis.  The  source  of  immune-bodies  is,  in  all 
probability,  also  the  leucocytes,  as  they  are  specially  abundant 
in  organs  rich  in  these  cells  —  spleen,  lymphatic  glands,  etc. ; 
here  again  the  mononuclear  leucocytes  are  probably  the  source 
of  the  immune-bodies  concerned  in  haemolysis,  the  polymorpho- 
nuclear  leucocytes  the  source  of  those  concerned  in  bacteri- 
olysis. Although  the  immune-bodies  are  usually  set  free  in  the 
serum,  this  is  not  always  the  case ;  sometimes  they  are  contained 
in  the  cells,  and  this  probably  occurs  when  there  is  a  high  degree 
of  active  immunity  against  bacteria  without  the  serum  having 
an  antibacterial  action.  In  this  way  the  facts  of  immunity  can 
be  explained  so  far  as  these  concern  the  destruction  of  bacteria. 

MetchnikofT s  work  has  less  direct  bearing  on  the  produc- 
tion of  antitoxins.  He  admits  the  fixation  of  the  toxin  by  the 
antitoxin  to  form  a  neutral  compound,  and  he  apparently  con- 
siders that  leucocytes  may  also  be  concerned  in  the  production 
of  antitoxins.  Apart,  however,  from  antitoxin  formation,  he  con- 
siders the  acquired  resistance  of  the  cells  themselves  of  high 
importance  in  toxin  immunity. 

When  we  consider  Metchnikorf's  theory  as  thus  extended 
to  cover  recently  established  facts,  it  must  be  admitted  that  it 
affords  a  rational  explanation  of  a  considerable  part  of  the 
subject,  provided  that  the  changes  in  the  chemiotactic  phe- 
nomena during  immunisation  are  fully  elucidated.  It,  however, 
does  not  afford  an  explanation  of  the  multiplicity  and  speci- 
ficity of  antitoxins  as  Ehrlich's  does ;  on  the  other  hand,  it  is 


496  IMMUNITY.  / 

more  concerned  with  the  cells  of  the  body  as  destroyers  or 
digesters  of  bacteria.  As  regards  the  subject  of  antibacterial 
sera,  the  results  of  these  two  workers  may  be  said  to  be  in 
harmony  in  some  of  the  fundamental  conceptions.  And  it  is 
of  interest  to  note  that  Metchnikoff,  starting  with  the  phe- 
nomena of  intracellular  digestion,  has  arrived  at  the  giving  off 
of  specific  ferments  by  phagocytes  ;  whilst  Ehrlich,  from  his 
first  investigations  on  the  constitution  of  toxins,  has  arrived  at 
an  explanation  of  antitoxins  and  immune-bodies  also  with  a 
theory  of  cell-nutrition  as  its  basis.  Within  the  last  few  years 
marked  progress  has  thus  been  made  towards  the  establishment 
of  the  fundamental  laws  of  immunity. 

NATURAL    IMMUNITY. 

We  have  placed  the  consideration  of  this  subject  after  that 
of  acquired  immunity,  as  the  latter  supplies  facts  which  indicate 
in  what  direction  an  explanation  of  the  former  may  be  looked 
for.  There  may  be  said  to  be  two  main  facts  with  regard  to 
natural  immunity.  The  first  is,  that  there  is  a  large  number  of 
bacteria  —  the  so-called  non-pathogenic  organisms  —  which  are 
practically  incapable,  unless  perhaps  in  very  large  doses,  of 
producing  pathogenic  effects  in  any  animal ;  when  these  are 
introduced  into  the  body,  they  rapidly  die  out.  This  fact  accord- 
ingly shows  that  the  animal  tissues  generally  have  a  remarkable 
power  of  destroying  living  bacteria.  The  second  fact  is,  that 
there  are  other  bacteria  which  are  very  virulent  to  some  species 
of  animals,  whilst  they  are  almost  harmless  to  other  species ; 
the  anthrax  bacillus  may  be  taken  as  an  example.  Now  it  is 
manifest  that  natural  immunity  against  such  an  organism  might 
be  due  to  a  special  powdr  possessed  by  an  animal  of  destroying 
the  organisms  when  introduced  into  its  tissues.  It  might  also, 
however,  be  due  to  an  insusceptibility  to,  or  power  of  neutral- 
ising, the  toxins  of  the  organism.  For  the  study  of  the  various 
diseases  shows  that  the  toxins  (in  the  widest  sense)  are  the 
weapons  by  which  morbid  changes  are  produced,  and  that  toxin- 
formation  is  a  property  common  to  all  pathogenic  bacteria. 
There  is,  moreover,  no  such  thing  known  as  a  bacterium  multi- 
plying in  the  living  tissues  without  producing  local  or  general 
changes,  though,  theoretically,  there  might  be.  We  may  infer 
from  this  that  if  the  toxins  are  completely  neutralised  or  ren 


NATURAL   IMMUNITY. 


497 


dered  powerless  in  the  case  of  any  animal,  that  animal  will  be 
immune  against  the  particular  organism.  This  is  also  borne 
out  by  the  fact  that  immunity  against  a  particular  organism  can 
be  artificially  obtained  by  injections  of  the  toxins  of  that  organ- 
ism. As  a  matter  of  fact,  however,  natural  immunity  is  in  most 
cases  one  against  infection,  i.e.  consists  in  a  power  possessed  by 
the  animal  body  of  destroying  the  living  bacteria  when  intro- 
duced into  its  tissues :  such  a  power  may  exist  though  the 
animal  is  still  susceptible  to  the  separated  toxins.  We  shall 
now  look  at  these  two  factors  separately. 

i.  Variations  in  Natural  Bactericidal  Powers. — The  fun- 
damental fact  here  is  that  a  given  bacterium  may  be  rapidly 
destroyed  in  one  animal,  whereas  in  another  it  may  rapidly 
multiply  and  produce  morbid  effects.  The  special  powers  of 
destroying  organisms  in  natural  immunity  have  been  ascribed  to 
(a)  phagocytosis,  and  (b)  the  action  of  the  serum. 

(a)  The  chief  factors  with  regard  to  phagocytosis  have  been 
given  above.  The  bacteria  in  a  naturally  immune  animal,  for 
example,  the  anthrax  bacillus  in  the  tissues  of  the  white  rat,  are 
undoubtedly  taken  up  in  large  numbers  and  destroyed  by  the 
phagocytes,  whereas  in  a  susceptible  animal  this  only  occurs  to 
a  small  extent ;  and  Metchnikoff  has  shown  that  they  are  taken 
up  in  a  living  condition,  and  are  still  virulent  when  tested  in 
a  susceptible  animal.  The  presence  or  absence  of  positive 
chemiotaxis  is  here  also  of  great  importance.  The  question, 
however,  is  whether  these  differences  in  chemiotaxis  are  not 
themselves  capable  of  explanation.  If  they  are,  then  the 
phagocytosis  per  se  is  rather  the  evidence  of  the  presence  of 
immunity  than  its  real  essence.  An  observation  of  Ehrlich's  on 
haemolytic  sera  is  somewhat  suggestive  in  this  connection.  The 
sera  of  some  animals  possess  naturally,  as  above  stated,  a 
haemolytic  action  on  the  blood  corpuscles  of  others,  and  in  the 
cases  studied  Ehrlich  found  that  this  was  not  due  to  complement 
(alexine)  alone,  but  to  complement  aided  by  an  intermediate 
body  (Zwischenkorper),  which  behaves  in  an  analogous  way  to 
the  immune-body  of  an  anti-serum.  As  already  pointed  out, 
bactericidal  action  closely  corresponds  with  haemolytic  action, 
and  it  is  quite  possible  that  in  a  naturally  immune  animal  some 
intermediate  substance  may  be  present  which  combines  with 
the  bacteria  and  thus  produces  some  change  which  is  evidenced 

2  K 


498  IMMUNITY. 

by  their  exerting  a  positive  chemiotaxis  on  the  leucocytes. 
Variations  in  phagocytic  activity  no  doubt  are  found  to  corre- 
spond more  or  less  closely  with  the  degree  of  immunity  present. 
(b)  When  it  had  been  shown  that  normal  serum  possessed 
bactericidal  powers  against  different  organisms,  the  question 
naturally  arose  as  to  whether  this  bactericidal  power  varied  in 
different  animals  in  proportion  to  the  natural  immunity  enjoyed 
by  them.  The  earlier  experiments  of  Behring  appeared  to  give 
grounds  for  the  belief  that  this  was  the  case.  He  found,  for 
example,  that  the  serum  of  the  white  rat,  which  has  a  remark- 
able immunity  to  anthrax,  had  greater  bactericidal  powers  than 
that  of  other  animals  investigated.  He  found  also  that  the 
serum  of  guinea-pigs  immunised  against  the  vibrio  Metchnikovi 
had  a  bactericidal  action,  whereas  in  that  of  susceptible  animals 
no  such  action  was  found.  Further  investigation,  however,  has 
shown  that  these  are  not  examples  of  a  general  law,  and  that 
this  bactericidal  action  of  the  serum  does  not  vary  pari  passu 
with  the  degree  of  immunity.  The  bactericidal  action  of  the 
serum  was  specially  studied  by  Nuttall,  and  later  by  Buchner 
and  Hankin,  who  believe  that  the  serum  owes  its  power  to 
certain  substances  in  it  derived  from  the  spleen,  lymphatic 
glands,  thymus,  and  other  tissues  rich  in  leucocytes.  To 
these  substances  Buchner  gave  the  name  of  alexines ;  as  al- 
ready explained,  they  correspond  with  MetchnikofP s  cystases 
and  Ehrlich's  complements.  These  substances  are  somewhat 
unstable  compounds,  and  are  destroyed  by  the  action  of  light, 
and  also  by  a  temperature  of  60°  C.  They  can  be  precipitated 
by  alcohol  and  by  ammonium  sulphate,  and  correspond  in  their 
general  behaviour  with  enzymes  or  unorganised  ferments.  Re- 
garding the  existence  in  the  serum  of  bactericidal  substances 
which  >are  very  easily  destroyed  by  heat  there  can  be  no  doubt, 
but  their  properties  can  only  be  studied  outside  the  body,  and  it 
must  not  be  assumed  that  the  serum  in  such  conditions  has  al- 
ways the  same  property  as  in  the  living  body.  In  some  cases, 
for  example,  the  bactericidal  power  of  the  serum  in  vitro  has 
been:  found  to  be  considerable,  while  the  animal  has  no  immu- 
nity. In  such  a  case  Metchnikoff  says  that  there  occurs  in  the 
living  body  no  liberation  of  alexines  by  the  phagocytes,  and 
hence  no  bactericidal  action  such  as  occurs  when  the  blood  is 
shed.  Variations  in  bactericidal  power  of  the  serum  as  tested 


VARIATIONS   IN    SUSCEPTIBILITY   TO   TOXINS.          499 

in  vitro,  therefore,  do  not  explain  the  presence  or  absence  of 
natural  immunity  against  a  living  bacterium. 

2.  Variations  in  Natural  Susceptibility  to  Toxins. — We  must 
here  start  with  the  fundamental  fact,  incapable  of  explanation, 
that  toxicity  is  a  relative  thing,  or  in  other  words,  that  different 
animals  have  different  degrees  of  resistance  or  non-suscepti- 
bility to  toxic  bodies.  In  every  case  a  certain  dose  must  be 
reached  before  effects  can  be  observed,  and  up  to  that  point 
the  animal  has  resistance.  This  natural  resistance  is  found  to 
present  very  remarkable  degrees  of  variation  in  different  ani- 
mals. The  great  resistance  of  the  common  fowl  to  the  toxin 
of  the  tetanus  bacillus  may  be  here  mentioned;  the  high  re- 
sistance of  the  pigeon  to  morphia  is  a  striking  example  in  the 
case  of  vegetable  poisons.  This  variation  in  resistance  to  toxins 
applies  also  to  those  which  produce  local  effects,  as  well  as  to 
those  which  cause  symptoms  of  general  poisoning.  Instances 
of  this  are  furnished,  for  example,  by  the  vegetable  poisons 
ricin  and  abrin,  by  the  snake  poisons,  and  by  bacterial  toxins 
such  as  that  of  diphtheria.  We  must  take  this  natural  resist- 
ance for  granted,  though  it  is  possible  that  ere  long  it  will  be 
explained. 

According  to  Ehrlich's  view  of  the  constitution  of  toxins,  it 
might  be  due  to  the  want  of  combining  affinity  between  the 
tissue  cells  and  the  haptophorous  group  of  the  toxin ;  or,  on  the 
other  hand,  supposing  this  affinity  to  exist,  it  might  be  due  to 
an  innate  non-susceptibility  to  the  action  of  the  toxophorous 
group.  Certain  investigations  have  been  made  in  order  to  de- 
termine the  combining  affinity  of  the  nervous  system  of  the 
fowl  with  tetanus  toxin,  as  compared  with  that  obtaining  in  a 
susceptible  animal,  but  the  results  have  been  somewhat  contra- 
dictory. Accordingly,  a  general  statement  on  this  point  cannot 
at  present  be  made. 

At  present,  therefore,  the  facts  of  natural  immunity  cannot 
be  fully  explained.  In  some  cases  the  insusceptibility  to  toxic 
substances  may  explain  the  degrees  of  immunity  possessed  by 
different  animals,  whilst  in  others  immunity  is  due  to  special 
bactericidal  powers  possessed  by  them.  What  these  bacteri- 
cidal powers  really  are  cannot  be  explained  on  any  single  the- 
ory. A  vital  activity  of  the  tissues  and  fluids  is,  no  doubt, 
brought  about  by  the  presence  of  the  bacteria,  and  this  cannot 


500  IMMUNITY. 

be  fully  imitated  in  experiments  outside  the  body.  The  facts 
given  above  with  regard  to  the  action  of  antibacterial  serum, 
show  how  complicated  a  matter  the  bactericidal  process  may  be. 
Further,  in  natural  immunity  a  direct  killing  of  the  organisms 
by  the  fluids  of  the  serum  is  not  necessary.  It  may  be  sufficient 
that  their  growth  is  prevented,  so  that  they  ultimately  die  out 
or  are  taken  up  by  the  phagocytes. 


APPENDIX   A. 

SMALLPOX   AND   VACCINATION. 

SMALLPOX  is  a  disease  to  which  much  study  has  been  devoted,  owing, 
on  the  one  hand,  to  the  havoc  which  it  formerly  wrought  among  the 
nations  of  Europe  —  a  havoc  which  at  the  present  day  it  is  difficult  to 
realise,  —  and  on  the  other  hand,  to  the  controversies  which  have  arisen 
in  connection  with  the  active  immunisation  against  it  introduced  by 
Jenner.  Though  there  is  little  doubt  that  a  contagium  vivum  is  con- 
cerned in  its  occurrence,  the  etiological  relationship  of  any  particular 
organism  to  smallpox  has  still  to  be  proved ;  and  with  regard  to  Jen- 
nerian  vaccination,  it  is  only  the  advance  of  bacteriological  knowledge 
which  is  now  enabling  us  to  understand  the  principles  which  underlie 
the  treatment,  and  which  is  furnishing  methods  whereby,  in  the  near 
future,  the  vexed  questions  concerned  will  probably  be  satisfactorily  set- 
tled. We  cannot  here  do  more  than  touch  on  some  of  the  results  of 
investigation  with  regard  to  the  disease. 

Jennerian  Vaccination.  —  Up  to  Jenner's  time  the  only  means  adopted 
to  mitigate  the  disease  had  been  by  inoculation  (by  scarification)  of 
virus  taken  from  a  smallpox  pustule,  especially  from  a  mild  case.  By 
this  means  it  was  shown  that  in  the  great  majority  of  cases  a  mild  form 
of  the  disease  was  originated.  It  had  previously  been  known  that  one 
attack  of  the  disease  protected  against  future  infection,  and  that  the 
mild  attack  produced  by  inoculation  also  had  this  effect.  This  inocula- 
tion method  had  long  been  practised  in  various  parts  of  the  world,  and 
had  considerable  popularity  all  over  Europe  during  the  eighteenth  cen- 
tury. Its  disadvantage  was  that  the  resulting  disease,  though  mild,  was 
still  infectious,  and  thus  might  be  the  starting-point  of  a  virulent  form 
among  unprotected  persons.  Jenner's  discovery  was  published  when 
inoculation  was  still  considerably  practised.  It  was  founded  on  the 
popular  belief  that  those  who  had  contracted  cowpox  from  an  affected 
animal  were  insusceptible  to  subsequent  infection  from  smallpox.  In 
the  horse  there  occurs  a  disease  known  as  horsepox,  especially  tending 
to  arise  in  wet  cold  springs,  which  consists  in  an  inflammatory  condition 
about  the  hocks,  giving  rise  to  ulceration.  Jenner  believed  that  the 
matter  from  these  ulcers,  when  transferred  by  the  hands  of  men  who 

501 


502  APPENDIX   A. 

dressed  the  sores  to  the  teats  of  cows-subsequently  milked  by  them,  gave 
:rise  to  cowpox  in  the  latter.  This  disease  was  thus  identical  with  horse- 
pox,  in  epidemics  of  which  it  had  its  origin.  Jenner  was,  however, 
probably  in  error  in  confounding  horsepox  with  another  disease  of 
horses,  namely,  "  grease."  Cowpox  manifests  itself  as  a  papular  eruption 
on  the  teats ;  the  papules  become  pustules ;  their  contents  dry  up  to 
form  scabs,  or  more  or  less  deep  ulcers  are  formed  at  their  sites.  From 
such  a  lesion  the  hands  of  the  milkers  may  become  infected  through 
abrasions,  and  a  similar  local  eruption  occurs,  with  general  symptoms  in 
the  form  of  slight  fever,  malaise,  and  loss  of  appetite.  It  is  this  illness, 
which,  according  to  Jenner,  gives  rise  to  immunity  from  smallpox  infec- 
tion. He  showed  experimentally  that  persons  who  had  suffered  from 
such  attacks  did  not  react  to  inoculation  with  smallpox,  and  further,  that 
persons  to  whom  he  communicated  cowpox  artificially,  were  similarly 
immune.  The  results  of  Jenner's  observations  and  experiments  were 
published  in  1 798  under  the  title  An  Inquiry  info  the  Causes  and  Effects 
of  the  Variola  Vaccines.  Though  from  the  first  Jennerian  vaccination 
had  many  opponents,  it  gradually  gained  the  confidence  of  the  unpreju- 
diced, and  became  extensively  practised  all  over  the  world,  as  it  is  at 
the  present  day. 

The  evidence  in  favour  of  vaccination  is  very  strong.  There  is  no 
doubt  that  inoculation  with  lymph  properly  taken  from  a  case  of  cow- 
pox  can  be  maintained  with  very  little  variation  in  strength  for  a  long 
time  by  passage  from  calf  to  calf,  and  such  calves  are  now  the  usual 
source  of  the  lymph  used  for  human  vaccination.  When  lymph  derived 
from  them  is  used  for  the  latter  purpose,  immunity" against  smallpox  is 
conferred  on  the  vaccinated  individual.  It  has  been  objected  that  some 
of  the  lymph  which  has  been  used  has  been  derived  from  calves  inocu- 
lated, not  with  cowpox,  but  with  human  smallpox.  It  is  possible  that 
this  may  have  occurred  in  some  of  the  strains  of  lymph  in  use  shortly 
after  the  publication  of  Jenner's  discovery,  but  there  is  no  doubt  that 
most  of  the  strains  at  present  in  use  have  been  derived  originally  from 
cowpox.  The  most  striking  evidence  in  favour  of  vaccination  is  derived 
from  its  effects  among  the  staffs  of  smallpox  hospitals,  for  here,  in 
numerous  instances,  it  is  only  the  unvaccinated  individuals  who  have 
contracted  the  disease.  While  vaccination  is  undoubtedly  efficacious  in 
protecting  against  smallpox,  Jenner  was  wrong  in  supposing  that  a  vac- 
cination in  infancy  afforded  protection  for  more  than  a  certain  number 
of  years  thereafter.  It  has  been  noted  in  smallpox  epidemics  which 
have  occurred  since  the  introduction  of  vaccination,  that  whereas  young 
unprotected  subjects  readily  contract  the  disease,  those  vaccinated  as 
Infants  escape  more  or  less  till  after  the  thirteenth  to  the  fifteenth  years. 
It  has  become,  therefore,  more  and  more  evident  that  revaccination  is 


SMALLPOX   AND   VACCINATION.  503 

necessary  if  immunity  is  to  continue;  and  where  this  is ;  done  in  any 
population,  smallpox  becomes  a  rare  disease,  as  has  happened  in  the 
German  army,  where  the  mortality  is  practically  nil.  The  whole  ques- 
tion of  the  efficacy  of  vaccination  was  investigated  in  Great  Britain  in 
1896  by  a  Royal  Commission,  whose  general  conclusions  were  as  fol- 
lows. Vaccination  diminishes  the  liability  to  attack  by  smallpox,  and 
when  the  latter  does  occur,  the  disease  is  milder  and  less  fatal.  Protec- 
tion against  attack  is  greatest  during  nine  or  ten  years  after  vaccination. 
It  is  still  efficacious  for  a  further  period  of  five  years,  and  possibly  never 
wholly  ceases.  The  power  of  vaccination  to  modify  attack  outlasts  its 
power  wholly  to  ward  it  off.  Revaccination  restores  protection,  but  this 
operation  must  be  from  time  to  time  repeated.  Vaccination  is  benefi- 
cial according  to  the  thoroughness  with  which  it  is  performed. 

The  Relationship  of  Smallpox  (Variola)  to  Cowpox  (Vaccinia) .  — 
This  is  the  question  regarding  which,  since  the  introduction  of  vaccina- 
tion, the  greatest  controversy  has  taken  place  ;  a  subsidiary  point  has 
been  the  inter-relationships  within  the  group  of  animal  diseases  which 
includes  cowpox,  horsepox,  sheep-pox,  and  cattle-plague.  With  refer- 
ence to  smallpox  and  cowpox  the  problem  has  been,  Are  they  identical 
or  not  ?  There  is  no  doubt  that  cowpox  can  be  communicated  to  man, 
in  whom  it  produces  the  eruption  limited  to  the  point  of  inoculation, 
and  the  slight  general  symptoms'which.  vaccination  with  calf  lymph  has 
made  familiar.  Apparently  against  the  view  that  cowpox  is  a  modified 
smallpox  are  the  facts  that  it  never  reproduces  in  man  a  general  erup- 
tion, and  that  the  local  eruption  is  only  infectious  when  matter  from  it  is 
introduced  into  an  abrasion.  The  loss  of  infectiveness  by  transmission 
through  the  body  of  a  relatively  insusceptible  animal  is  a  condition  of 
which  we  have  already  seen  many  instances  in  other  diseases,  and  the 
uniformity  of  the  type  of  the  affection  resulting  from  vaccination  with 
calf  lymph  finds  a  parallel  in  such  a  disease  as  hydrophobia,  where, 
after  passage  through  a  series  of  monkeys,  a  virus  of  attenuated  but 
constant  virulence  can  be  obtained.  We  have  seen  that  there  are  good 
grounds  for  believing  that  the  virus  of  calf  lymph  confers  immunity 
against  human  smallpox.  In  considering  the  relationships  of  cowpox 
and  smallpox,  this  is  an  important  though  subsidiary  point  ;  for  at 
present  it  is  questionable  whether  there  are  any  well-authenticated 
instances  of  one  disease  having  the  capacity  of  conferring  immunity 
against  another.  The  most  difficult  question  in  this  connection  is  what 
happens  when  inoculations  of  smallpox  matter  are  made  on  cattle. 
Chauveau  denies  that  in  such  circumstances  cowpox -is  obtained.  He, 
however,  only  experimented  on  adult  cows.  The  transformation  has 
been  accomplished  by  many  observers,  including,  in  Britain,  Simp- 
son, Klein,  Hime,  and  Copeman.  The  general  result  of  these  experi- 


504  APPENDIX  A. 

merits  has  been  that  if  a  series  of  calves  is  inoculated  with  variolous 
matter,  in  the  first  there  may  not  be  much  local  reaction,  though  red- 
ness and  swelling  appear  at  the  point  of  inoculation,  and  some  general 
symptoms  manifest  themselves.  On  squeezing  some  of  the  lymph  from 
such  reaction  as  occurs,  and  using  it  to  continue  the  passages  through 
other  calves,  after  a  very  few  transfers  a  local  reaction  indistinguishable 
from  that  caused  by  cowpox  lymph  generally  takes  place,  and  the  ani- 
mals are  now  found  to  be  immune  against  the  latter.  Not  only  so,  but 
on  using  for  human  vaccination  the  lymph  from  such  variolated  calves, 
results  indistinguishable  from  those  produced  by  vaccine  lymph  are 
obtained,  and  the  transitory  illness  which  follows,  unlike  that  produced 
in  man  by  inoculation  with  smallpox  lymph,  is  no  longer  infectious.  In 
fact  many  of  the  strains  of  lymph  in  use  in  Germany  at  present  have 
been  derived  thus  from  the  variolation  of  calves.  The  criticism  of 
these  experiments  which  has  been  offered,  namely,  that  since  many  of 
them  were  performed  in  vaccine  establishments,  the  calves  were  prob- 
ably at  the  same  time  infected  with  vaccine,  is  not  of  great  weight,  as  in 
all  the  recent  cases  at  least,  very  elaborate  precautions  have  been 
adopted  against  such  a  contingency.  And  at  any  rate  it  would  be 
rather  extraordinary  that  this  accident  should  happen  to  occur  in  every 
case.  We  can,  therefore,  say  that  at  present  there  is  the  very  strongest 
ground  for  holding  not  only  that  vaccinia  confers  immunity  against 
variola,  but  that  variola  confers  immunity  against  vaccinia.  The  experi- 
mentum  crucis  for  establishing  the  identity  of  the  two  diseases  would  of 
course  be  the  isolation  of  the  same  micro-organism  from  both,  and  the 
obtaining  of  all  the  results  just  detailed  by  means  of  pure  cultures  or 
the  products  of  such.  In  the  absence  of  this  evidence  we  are  at 
present  justified  in  considering  that  there  is  strong  reason  for  believing 
that  vaccinia  and  variola  are  the  same  disease,  and  that  the  differences 
between  them  result  from  the  relative  susceptibilities  of  the  two  species 
of  animals  in  which  they  naturally  occur. 

With  regard  to  the  relation  of  cowpox  to  horsepox,  it  is  extremely 
probable  that  they  are  the  same  disease.  Some  epidemics  of  the  former 
have  originated  from  the  horse,  but  in  other  cases  such  a  source  has  not 
been  traced.  Cattle-plague,  from  the  clinical  standpoint,  and  also  from 
that  of  pathological  anatomy,  resembles  very  closely  human  smallpox. 
Though  each  of  the  two  diseases  is  extremely  infectious  to  its  appro- 
priate animal,  there  is  no  record  of  cattle-plague  giving  rise  to  smallpox 
in  man  or  vice  versa.  When  matter  from  a  cattle-plague  pustule  is 
inoculated  in  man,  a  pustule  resembling  a  vaccine  pustule  occurs,  and 
further,  the  individual  is  asserted  to  be  now  immune  to  vaccination  ; 
but  vaccination  of  cattle  with  cowpox  lymph  offers  no  protection  against 
cattle-plague,  though  some  have  looked  on  the  latter  as  merely  a  malig- 


SMALLPOX   AND   VACCINATION. 


505 


nant  cowpox.  Sheep-pox  also  has  many  clinical  and  pathological  anal- 
ogies with  human  smallpox,  and  facts  as  to  its  relation  to  cowpox 
vaccination  similar  to  those  observed  in  cattle-plague  have  been,  re- 
ported. Smallpox,  cowpox,  cattle-plague,  horsepox,  and  sheep-pox,  in 
short,  constitute  an  interesting  group  of  analogous  diseases,  of  the  true 
relationships  of  which  to  one  another  we  are,  however,  still  ignorant. 

Micro-organisms  associated  with  Smallpox.  —  Burdon  Sanderson  was 
among  the  first  to  show  that  in  vaccine  lymph  there  were  certain  bodies 
which  he  recognised  as  bacteria.  Since  then  numerous  observations 
have  been  made  as  to  the  occurrence  of  such  in  matter  derived  from 
variolous  and  vaccine  pustules.  In  especially  the  later  stages  of  the 
latter,  many  of  the  pyogenic  organisms  are  always  present,  e.g.  staphylo- 
coccus  aureus  and  staphylococcus  cereus  flavus,  and  many  of  the  ordinary 
skin  saprophytes  also  are  often  present,  but  no  organism  has  ever  been 
isolated  which-  on  transference  to  animals  has  been  shown  to  have  any 
specific  relationship  to  the  disease.  Klein,  and  also,  independently, 
Copeman,  have  observed  an  organism  in  lymph  taken  from  a  vaccine 
pustule  in  a  calf  on  the  fifth  and  sixth  days/ in  human  vaccine  lymph  on 
the  eighth  day,  and  in  lymph  from  a  smallpox  pustule  on  the  fourth  day. 
To  demonstrate  the  bacilli,  cover-glass  films  are  dried  and  placed  for 
five  minutes  in  acetic  acid  (i  in  2),  washed  in  distilled  water,  dried,  and 
placed  in  alcoholic  gentian-violet  for  from  twenty-four  to  forty- eight 
hours,  after  which  they  are  washed  in  water  and  mounted.  Copeman 
and  Kent  also  found  the  bacilli  in  sections  of  vaccine  pustules  stained 
by  LorHer's  methylene-blue,  or  by  Gram's  method.  The  organisms  are 
.4  to  .8  fji  in  length,  and  one-third  to  a  half  of  this  in  thickness.  They 
are  generally  thinner  and  stain  better  at  the  ends  than  at  the  middle. 
They  occur  in  groups  of  from  three  to  ten  in  both  the  lymph  and  the 
tissues.  In  the  centre  of  their  protoplasm  there  is  often  a  clear  globule, 
which  is  looked  on  as  a  spore.  They  have  hitherto  resisted  the  ordi- 
nary isolation  methods,  a  fact  which  is  rather  in  favour  of  their  non- 
saprophytic  nature.  By  inoculating  fresh  eggs  with  the  crusts  of 
smallpox  pustules  Copeman  has,  however,  obtained  a  growth  of  a  bacillus 
resembling  that  found  by  him  in  the  tissues.  Though  sub-cultures  on 
ordinary  media  have  been  obtained,  the  pathogenic  effects  of  these  have 
not  been  fully  investigated,  and  thus  the  identity  of  this  bacillus  with 
that  seen  in  the  tissues  is  not  proved.  The  facts  that  the  latter  is  one 
hitherto  not  recognised  microscopically,  that  it  exists  in  the  pustules,  the 
contents  of  which  are  probably  the  means  by  which  the  disease  naturally 
spreads,  that  it  resists  artificial  cultivation,  that  the  possession  by  it  of 
spores  explains  some  of  the  characteristics  of  vaccine  lymph  (resistance 
to  drying,  etc.),  are,  however,  of  interest  from  the  point  of  view  of  the 
possible  etiological  relationship  of  the  bacillus  to  the  disease. 


506  APPENDIX   A. 

Protozoa  as  Causative  Agents.  —  Van  der  Loeff  and  L.  Pfeiffer  early 
drew  attention  to  the  presence  of  small  amoeboid  bodies  in  the  blood 
and  epithelial  cells  of  the  skin  and  mucous  membranes  of  persons  affected 
with  smallpox,  which  they  believed  to  be  the  cause  of  the  disease,  and 
considered  them  as  protozoa.  Later,  Guarnieri,  in  a  series  of  studies 
upon  rabbits  which  he  had  inoculated  in  the  cornea  with  active  vaccine 
lymph,  described  bodies  occurring  in  the  lesions  similar  to  those  of  Van 
der  Loeff  and  Pfeiffer.  He  further  showed  that  when  using  naturally 
inactive  lymph,  or  filtered  vaccine  lymph,  no  bodies  were  produced  in 
the  corneal  cells,  but  they  again  appeared  if  the  material  held  back  by 
the  filter  were  inoculated. 

Ruffer  and  Plimmer  describe  as  occurring  in  clear  vacuoles  in  the 
cells  of  the  rete  Malpighii  at  the  edge  of  the  pustule,  in  paraffin  sections 
of  vaccine  and  smallpox  pustules  carefully  hardened  in  alcohol,  and 
stained  by  the  Ehrlich-Biondi  mixture,  small  round  bodies  about  four 
times  the  size  of  a  staphylococcus  pyogenes,  coloured  red  by  the  acid 
fuchsin,  sometimes  with  a  central  part  stained  by  the  methyl-green. 
These  appear  to  multiply  by  simple  division,  and  in  the  living  condition 
exhibit  amoeboid  movement.  Similar  bodies  have  been  described  by 
Reed  in  the  blood  of  smallpox  patients  and  of  vaccinated  children  and 
of  calves. 

In  an  exhaustive  research  von  Wasielewski,  upon  a  larger  scale  repeating  Guar- 
nieri's  work,  largely  confirms  the  latter's  observations.  He  utilised  both  human  and 
calf  vaccine  lymphs,  and  in  one  series,  using  bacteria-free  calf  lymph,  he  was  able  to 
reproduce  the  peculiar  bodies  in  the  cornea  of  a  rabbit  with  the.  forty-eighth  transfer, 
passing  from  one  rabbit  to  another  throughout  the  series.  He  found  similar  bodies 
in  the  epithelial  cells  in  a  case  of  smallpox,  too.  His  conclusions  are  that  these 
bodies  are  neither  cell-inclusions  of  leucocytic  origin,  nor  degeneration  products  of 
the  epithelial  cells  themselves,  but,  with  Guarnieri,  believes  that  in  all  probability 
they  are  the  causative  factors  of  the  disease. 

Another  observer,  Gorini,  lays  great  stress  upon  the  presence  of  coccus-like  bodies 
free  and  in  the  cells  of  vaccine  vesicles  and  in  the  lymph,  which  occur  singly,  in  pairs, 
and  in  tetrads.  He  thinks  that  possibly  their  nature  is  bacterial,  and  that,  being 
absent  in  inactive  lymph  and  in  the  cells  of  ordinary  inflammatory  origin,  they 
possibly  may  be  the  cause  of  the  phenomena  of  vaccination.  Gorini  believes  that  the 
bodies  described  by  Guarnieri  and  von  Wasielewski  (cytoryctes  vaccince)  are  simply 
the  products  of  cell  degeneration. 

In  the  presence  of  such  interesting  but  divergent  findings,  it  is  plain 
that  more  precise  research  of  a  confirmatory  nature  is  required  before 
any  of  these  bodies  can  be  accepted  as  being  the  cause  of  smallpox  or 
vaccination. 

The  Nature  of  Vaccination.  —  As  we  are  ignorant  of  the  cause  of  small- 
pox, we  can  only  conjecture  what  the  nature  of  vaccination  is.  From 
what  we  know  of  other  like  processes,  however,  we  have  some  ground 


SMALLPOX   AND   VACCINATION.  507 

for  believing  that  it  consists  in  an  active  immunisation  by  means  of  an 
attenuated  form  of  the  causal  organism.  As  to  how  immunity  is  main- 
tained after  vaccination,  we  do  not  know  much.  Some,  including 
Beclere,  Chambon,  and  Menard  (who  jointly  investigated  the  subject), 
maintain  that  in  the  blood  of  vaccinated  animals  substances  exist  which, 
when  transferred  to  other  animals,  can  confer  a  certain  degree  of  passive 
immunity  against  vaccination,  and  which  have  also  a  degree  of  curative 
action  in  animals  already  vaccinated.  Beumer  and  Peiper,  on  the  other 
hand,  could  not  find  evidence  of  the  existence  of  such  bodies.  If  they 
do  exist,  we  cannot  as  yet  say  whether  they  are  antitoxic  or  antimicrobic. 


APPENDIX    B. 
HYDROPHOBIA. 

SYNONYMS  :  —  RABIES.     FRENCH,   LA   RAGE.     GERMAN,   LYSSA, 
DIE  HUNDSWUTH,  DIE  TOLLWUTH. 

Introductory.  —  Hydrophobia  is  an  infectious  disease  which  in 
nature  occurs  epidemically  chiefly  among  the  carnivora,  especially  in 
the  dog  and  the  wolf.  Infection  is  carried  by  the  bite  of  a  rabid  ani- 
mal or  by  a  wound  or  abrasion  being  licked  by  such.  The  disease  can 
be  transferred  to  other  species,  and  when  once  started  can  be  spread 
from  individual  to  individual  by  the  same  paths  of  infection.  Thus  it 
occurs  epidemically  from  time  to  time  in  cattle,  sheep,  horses,  and  deer, 
and  can  be  communicated  to  man ;  but  in  modern  times  at  least,  infec- 
tion practically  never  takes  place  from  man  to  man,  though  such  an 
occurrence  is  quite  possible. 

In  Western  Europe  the  disease  is  most  frequently  observed  in  the 
dog;  but  in  Eastern  Europe,  especially  in  Russia,  epidemics  among 
wolves  constitute  a  serious  danger  both  to  other  animals  and  to  man. 
All  the  manifestations  of  the  disease  point  to  a  serious  affection  of  the 
nervous  system  ;  but  inasmuch  as  symptoms  of  excitement  or  of  depres- 
sion may  predominate,  it  is  customary  to  describe  clinically  two  varieties 
of  rabies,  (i)  rabies  proper,  or  furious  rabies  (la  rage  vraie,  la  rage 
furieuse :  die  rasende  WutJi] ;  and  (2)  dumb  madness  or  paralytic  rabies 
(la  rage  mue ;  die  stille  Wuth).  The  disease,  however,  is  essentially  the 
same  in  both  cases.  In  the  dog  the  furious  form  is  the  more  common. 
After  a  period  of  incubation  of  from  three  to  six  weeks,  the  first  symptom 
noticed  is  a  change  in  the  animal's  aspect ;  it  becomes  restless,  it  snaps 
at  anything  which  it  touches,  and  tears  up  and  swallows  unwonted 
objects ;  it  has  a  peculiar  high-toned  bark.  Spasms  of  the  throat 
muscles  come  on,  especially  in  swallowing,  and  there  is  abundant  secre- 
tion of  saliva ;  its  supposed  special  fear  of  water  is,  however,  a  myth, 
— it  fears  to  swallow  at  all.  Gradually  convulsions,  paralysis,  and  coma 
come  on  ;  and  death  supervenes.  In  the  paralytic  form,  the  early  symp- 
toms are  the  same,  but  paralysis  appears  sooner.  The  lower  jaw  of  the 
animal  drops,  from  implication  of  the  elevator  muscles,  all  the  muscles 

508 


HYDROPHOBIA.  509 

of  the  body  become  more  or  less  weakened,  and  death  ensues  without 
any  very  marked  irritative  symptoms. 

In  man  the  incubation  period  after  infection  varies  from  fifteen  days 
to  seven  or  eight  months,  or  even  longer,  but  is  usually  about  forty  days. 
When  symptoms  of  rabies  are  about  to  appear,  certain  prodromata,  such 
as  pains  in  the  wound  and  along  the  nerves  of  the  limb  in  which  the 
wound  has  been  received,  may  be  observed.  To  this  succeeds  a  stage 
of  nervous  irritability,  during  which  all  the  reflexes  are  augmented  — 
the  victim  starting  at  the  slightest  sound,  for  example.  There  are 
spasms,  especially  of  the  muscles  of  deglutition  and  respiration,  and 
cortical  excitement  evidenced  by  delirium  may  occur.  On  this  follows 
a  period  in  which  all  the  reflexes  are  diminished,  weakness  and  paralysis 
are  observed,  convulsions  occur,  and  finally  coma  and  death  supervene. 
The  duration  of  the  acute  illness  is  usually  from  four  to  eight  days,  and 
•death  invariably  results.  The  existence  of  paralytic  rabies  in  man  has  been 
•denied  by  some,  but  it  undoubtedly  occurs.  This  is  usually  manifested 
by  paralysis  of  the  limb  in  which  the  infection  has  been  received,  and 
•of  the  neighbouring  parts ;  but  while  in  such  cases  this  is  often  the  first 
symptom  observed,  during  the  whole  of  the  illness  the  occurrence  of 
widespread  and  progressive  paralysis  is  the  outstanding  feature. 

The  Pathology  of  Hydrophobia.  —  In  hydrophobia,  as  in  tetanus,  to 
which  it  bears  more  than  a  superficial  resemblance,  the  appearances 
presented  in  the  nervous  system,  to  which  all  the  symptoms  are  natu- 
rally referred,  are  comparatively  unimportant.  On  naked-eye  examina- 
tion, congestions,  and,  it  may  be,  minute  haemorrhages  in  the  central 
nervous  system,  are  the  only  features  noticeable.  Microscopically, 
leucocytic  exudation  into  the  perivascular  lymphatic  spaces  in  the 
nerve  centres  has  been  observed,  and  in  the  cells  of  the  anterior  cornua 
of  the  grey  matter  in  the  'spinal  cord,  and  also  in  the  nuclei  of  the 
-cranial  nerves,  various  degenerations  have  been  described.  In  the  white 
matter,  especially  in  the  posterior  columns,  swelling  of  the  axis  cylinders 
and  breaking  up  of  the  myeline  sheaths  have  been  noted,  and  similar 
changes  occur  also  in  the  spinal  nerves,  especially  of  the  part  of  the 
body  through  which  infection  has  come.  In  the  nervous  system  also 
some  have  seen  minute  bodies  which  they  have  considered  to  be  cocci, 
but  that  they  are  really  such  there  is  no  evidence.  Nelis  and  van 
Gehuchten  have  drawn  attention  to  early  and  well-marked  changes 
occurring  in  the  peripheral,  cerebral,  and  sympathetic  ganglia,  especially 
in  the  intervertebral  ganglia  and  in  the  plexiform  ganglia  of  the  pneu- 
mogastric  nerve,  consisting  in  the  invasion  and  ultimate  destruction  of 
the  nerve-cell  protoplasm  by  newly  formed  cells  derived  from  the 
capsular  membrane.  The  lesions  are  most  perceptible  upon  the  death 
of  the  animal,  although  they  can  be  made  out  if  the  animal  be  killed 


510  APPENDIX   B. 

after  symptoms  of  the  disease  are  strongly  developed.  The  work  of  these 
observers  has  been  confirmed  in  America  by  Ravenel  and  McCarthy, 
but  Spiller  has  noted  similar  changes  in  man  in  a  case  of  Landry's 
paralysis  and  does  not  think  the  lesion  specific  of  rabies.  Nevertheless, 
given  a  case  of  "  street-rabies,"  the  diagnosis  can  be  made  with  cer- 
tainty much  earlier  than  by  the  inoculation  method.  The  changes  in 
the  other  parts  of  the  body  are  unimportant. 

Experimental  pathology  confirms  the  view  that  the  nervous  system 
is  the  centre  of  the  disease  by  finding  in  it  a  special  concentration  of 
what,  from  want  of  a  more  exact  term,  we  must  call  the  hydrophobic 
virus.  Earlier  inoculation  experiments  made  by  subcutaneous  injection 
of  material  from  various  parts  of  animals  dead  of  rabies  had  not  given 
uniform  results,  as,  whatever  was  the  source  of  the  material,  the  disease 
was  not  invariably  produced.  Pasteur's  first  contribution  to  the  subject 
was  to  show  that  the  most  certain  method  of  infection  was  by  inserting 
the  infective  matter  beneath  the  dura  mater.  He  found  that  in  the  case 
of  any  animal  or  man  dead  of  the  disease,  injection  by  this  method  of 
emulsions  of  any  part  of  the  central  nervous  system,  of  the  cerebro-spinal 
fluid,  or  of  the  saliva,  invariably  give  rise  to  rabies,  and  also  that  the 
natural  period  of  incubation  was  shortened.  Further,  the  identity  of 
the  furious  and  paralytic  forms  was  proved,  as  sometimes  the  one,  some- 
times the  other,  was  produced,  whatever  form  had  been  present  in  the 
original  case.  Inoculation  into  the  anterior  chamber  of  the  eye  is  nearly 
as  efficacious  as  subdural  infection.  Infection  with  the  blood  of  rabic 
animals  does  not  reproduce  the  disease.  There  is  evidence,  however,, 
that  the  poison  also  exists  in  such  glands  as  the  pancreas  and  mamma. 
Subcutaneous  infection  with  part  of  the  nervous  system  of  an  animal 
dead  of  rabies  usually  gives  rise  to  the  disease. 

In  consequence  of  the  introduction  of  such  reliable  inoculation 
methods,  further  information  has  been  acquired  regarding  the  spread 
and  distribution  of  the  virus  in  the  body.  Gaining  entrance  by  the  in- 
fected wound,  it  early  manifests  its  affinity  for  the  nervous  tissues.  It 
reaches  the  central  nervous  system  chiefly  by  spreading  up  the  peripheral 
nerves.  This  can  be  shown  by  inoculating  an  animal  subcutaneously 
in  one  of  its  limbs,  with  virulent  material.  If  now  the  animal  be  killed 
before  symptoms  have  manifested  themselves,  rabies  can  be  produced 
by  subdural  inoculation  from  the  nerves  of  the  limb  which  was  infected. 
Further,  rabies  can  often  be  produced  from  such  a  case  by  subdural  in- 
fection with  the  part  of  the  spinal  cord  into  which  these  nerves  pass, 
while  the  other  parts  of  the  animal's  nervous  system  do  not  give  rise  to 
the  disease.  This  explains  how  the  initial  symptoms  of  the  disease 
(pains  along  nerves,  paralysis,  etc.)  so  often  appear  in  the  infected  part 
of  the  body,  and  it  probably  also  explains  the  fact  that  bites  in  such  richly 


HYDROPHOBIA.  5II 

nervous  parts  as  the  face  and  head  are  much  more  likely  to  be  followed 
by  hydrophobia  than  bites  in  other  parts  of  the  body.  Again,  injection 
into  a  peripheral  nerve,  such  as  the  sciatic,  is  almost  as  certain  a 
method  of  infection  as  injection  into  the  subdural  space,  and  gives  rise 
to  the  same  type  of  symptoms  as  injection  into  the  corresponding  limb. 
Intravenous  injection  of  the  virus,  on  the  other  hand,  differs  from  the 
other  modes  of  infection  in  that  it  more  frequently  gives  rise  to  paralytic 
rabies.  This  fact  Pasteur  explained  by  supposing  that  the  whole  of  the 
nervous  system  in  such  a  case  becomes  simultaneously  affected.  The 
virus  seems  to  have  an  elective  affinity  for  the  salivary  glands,  as  well  as 
for  the  nervous  system.  Roux  and  Nocard  found  that  the  saliva  of  the 
dog  became  virulent  three  days  before  the  first  appearance  of  symptoms 
of  the  disease. 

The  Virus  of  Hydrophobia.  — While  a  source  of  infection  undoubtedly 
occurs  in  all  cases  of  hydrophobia,  and  can  usually  be  traced,  all  attempts 
to  determine  the  actual  morbific  cause  have  been  unsatisfactory.  In 
this  connection  various  organisms  have  been  described  as  being  associ- 
ated with  the  disease.  Thus  Memmo  has  isolated  an  organism  which 
resembles  a  yeast,  but  which  he  places  amongst  the  blastomycetes,  and 
with  which  he  states  he  has  produced  both  types  of  rabies  in  rabbits  and 
dogs.  Bruschettini  also,  by  using  media  containing  brain  substance,  has 
grown  a  bacillus  having  some  resemblances  to  the  members  of  the  diph- 
theria group,  and  with  which  he  claims  to  have  produced  paralytic 
rabies  in  rabbits.  In  the  case  of  the  work  of  neither  of  these  observers 
has  there  been  confirmation  from  independent  sources,  and  in  neither 
case  is  there  evidence  of  the  crucial  test  having  been  applied,  namely, 
that  of  immunising  animals  against  the  ordinary  hydrophobic  virus  by 
means  of  pure  cultures  of  the  alleged  causal  organism.  With  regard  to 
other  possible  causal  agents,  Grigorjew  thinks  such  may  be  found  in 
a  protozoon  which  he  has  constantly  observed  after  inoculation  in  the 
cornea.  There  is  no  doubt  that  between  rabies  and  the  bacterial  dis- 
eases we  have  studied  there  are  at  every  point  analogies,  the  most  strik- 
ing being  the  protective  inoculation  methods  which  constitute  the  great 
work  of  Pasteur,  and  everything  points  to  a  micro-organism  being  the 
cause.  Judging  from  our  knowledge  of  similar  diseases  we  would 
strongly  suspect  that  it  is  actually  present  in  a  living  condition  in  the 
central  nervous  system,  the  saliva,  etc.,  which  yield  what  we  have  called 
the  hydrophobic  virus,  for  by  no  mere  toxin  could  the  disease  be  trans- 
mitted, through  a  series  of  animals,  as  we  shall  presently  see  can  be  done. 
The  resistance  of  the  virus  to  external  agents  varies.  Thus  a  nervous 
system  containing  it  is  virulent  till  destroyed  by  putrefaction ;  it  can  re- 
sist the  prolonged  application  of  a  temperature  of  from  —  10°  to  —  20° 
C.,  but,  on  the  other  hand,  it  is  rendered  non-virulent  by  one  hour's 


512  APPENDIX   B. 

exposure  at  50°  C.  Again,  its  potency  probably  varies  in  nature  accord- 
ing to  the  source.  Thus,  while  the  death-rate  among  persons  bitten  by 
mad  dogs  is  about  16  per  cent,  the  corresponding  death-rate  after  the 
bites  of  wolves  is  80  per  cent.  Here,  however,  it  must  be  kept  in  view 
that,  as  the  wolf  is  naturally  the  more  savage  animal,  the  number  and 
extent  of  the  bites,  i.e.  the  number  of  channels  of  entrance  of  the  virus 
into  the  body,  and  the  total  dose,  are  greater  than  in  the  case  of  persons 
bitten  by  dogs.  As  we  shall  see,  alterations  in  the  potency  of  the  virus 
can  certainly  be  effected  by  artificial  means. 

The  Prophylactic  Treatment  of  Hydrophobia.  —  Until  the  publica- 
tion of  Pasteur's  researches  in  1885,  the  only  means  adopted  to  prevent 
the  development  of  hydrophobia  in  a  person  bitten  by  a  rabid  animal, 
had  consisted  in  the  cauterisation  of  the  wound.  Such  a  procedure  was 
undoubtedly  not  without  effect.  It  has  been  shown  that  cauterisation 
within  five  minutes  of  the  infliction  of  a  rabic  wound  prevents  the  disease 
from  developing,  and  that  if  done  within  half  an  hour,  it  saves  a  propor- 
tion of  the  cases.  After  this  time,  cauterisation  only  lengthens  the 
period  of  incubation ;  but,  as  we  shall  see  presently,  this  is  an  extremely 
important  effect. 

The  work  of  Pasteur  has,  however,  revolutionised  the  whole  treat- 
ment of  wounds  inflicted  by  hydrophobic  animals.  Pasteur  started  with 
the  idea  that,  since  the  period  of  incubation  in  the  case  of  animals  in- 
fected subdurally  from  the  nervous  systems  of  mad  dogs  is  constant  in 
the  dog,  the  virus  has  been  from  time  immemorial  of  constant  strength. 
Such  a  virus,  of  what  might  be  called  natural  strength,  is  usually  referred 
to  in  his  works  as  the  virus  of  la  rage  des  rues,  in  the  writings  of  Ger- 
man authors  as  the  virus  of  die  Strasswuth.  Pasteur  found  on  inoculat- 
ing a  monkey  subdurally  with  such  a  virus,  and  then  inoculating  a  second 
monkey  from  the  first,  and  so  on  with  a  series  of  monkeys,  that  it  gradu- 
ally lost  its  virulence,  as  evidenced  by  lengthened  periods  of  incubation 
on  subdural  inoculation  of  dogs,  until  it  wholly  lost  the  power  of  pro- 
ducing rabies  in  dogs,  when  introduced  subcutaneously.  When  this 
point  had  been  attained,  its  virulence  was  not  diminished  by  further 
passage  through  the  monkey.  On  the  other  hand,  if  the  virus  of  la  rage 
des  rues  were  similarly  passed  through  a  series  of  rabbits  or  guinea-pigs, 
its  virulence  was  increased  till  a  constant  strength  (the  virus  fixe}  was 
attained.  Pasteur  had  thus  at  command  three  varieties  of  virus  —  that 
of  natural  strength,  that  which  had  been  attenuated,  and  that  which  had 
been  exalted.  He  further  found  that,  commencing  with  the  subcuta- 
neous injection  of  a  weak  virus  and  following  this  up  with  the  injection 
of  the  stronger  varieties,  he  could  ultimately,  in  a  very  short  time, 
immunise  dogs  against  subdural  infection  with  a  virus  which,  under 
ordinary  conditions,  would  certainly  have  caused  a  fatal  result.  He  also 


HYDROPHOBIA. 


513 


elucidated  the  fact  that  the  exalted  virus  contained  in  the  spinal  cords 
of  rabbits  such  as  those  referred  to  could  be  attenuated  so  as  no  longer 
to  produce  rabies  in  dogs  by  subcutaneous  injection.  This  was  done  by 
drying  the  cords  in  air  over  caustic  potash  (to  absorb  the  moisture),  the 
diminution  of  virulence  being  proportional  to  the  length  of  time  during 
which  the  cords  were  kept.  Accordingly,  by  taking  a  series  of  such  spinal 
cords  kept  for  various  periods  of  time,  he  was  supplied  with  a  series  of 
vaccines  of  different  strengths.  Pasteur  at  once  applied  himself  to  find 
whether  the  comparatively  long  period  of  incubation  in  man  could  not 
be  taken  advantage  of  to  "  vaccinate  "  him  against  the  disease  before  its 
gravest  manifestation  took  place.  The  following  is  the  record  of  the 
first  case  thus  treated.  The  technique  was  to  rub  up  in  a  little  sterile 
bouillon  a  small  piece  of  the  cord  used,  and  inject  _it  under  the  skin  by 
means  of  a  hypodermic  syringe.  The  first  injection  was  made  with  a 
very  attenuated  virus,  i.e.  a  cord  fourteen  days  old.  In  subsequent  in- 
jections the  strength  of  the  virus  was  gradually  increased,  as  shown  in  the 
table  :  — 

July    7,  1885,  9  A.M.,  cord  of  June  23,  i.e.  14  days  old. 


7 

„        6  P.M.                 „ 

25          „ 

12 

8 

„        9  A.M. 

27         „ 

II 

8- 

„        6  P.M. 

29         „ 

9 

9 

„     ii  A.M.,  cord  of  July 

I          J> 

8 

10 

»         »                   » 

3    » 

7 

ii 

5>                  »                                       99 

5         » 

6 

12 

»                  M                                      » 

7    » 

5 

13 

»»                  »                                      » 

9    „ 

4 

14 

»                  »                                      >» 

ii     „ 

3 

15 

»»                  »                                      » 

13    » 

2 

i6 

»                  >»                                       » 

15    » 

I 

day  old. 

The  patient  never  manifested  the  slightest  symptom  of  hydrophobia. 
Other  similarly  favourable  results  followed ;  and  this  prophylactic  treat- 
ment of  the  disease  quickly  gained  the  confidence  of  the  scientific  world, 
which  it  still  maintains.  (The  principle  is,  of  course,  the  same  as  in 
artificially  developing  a  high  degree  of  active  immunity  against  a  bac- 
terial infection.) 

The  only  modification  which  the  method  has  undergone  has  been  in  the  treat- 
ment of  serious  cases,  such  as  multiple  bites  from  wolves,  extensive  bites  about  the 
head,  especially  in  children,  cases  which  come  under  treatment  at  a  late  period  of 
the  incubation  stage,  and  cases  where  the  wounds  have  not  cicatrised.  In  such  cases 
the  stages  of  the  treatment  are  condensed.  Thus  on  the  first  day,  say  at  1 1  A.M.  and 
4  P.M.  and  9  P.M.,  cords  of  12,  10,  and  8  days  respectively  are  used  ;  on  the  second 
day,  cords  of  6,  4,  and  2  days  ;  on  the  third  day,  a  cord  of  I  day  ;  on  the  fourth  day, 
cords  of  8,  6,  and  4  days  ;  on  the  fifth,  cords  of  3  and  2  days  ;  on  the  sixth,  cords 
of  I  day  ;  and  so  on  for  ten  days.  In  each  case  the  average  dose  is  about  2  c.c. 
of  the  emulsion. 

2  I. 


514  APPENDIX   B. 

The  success  of  the  treatment  has  been  very  marked.  The  statistics  of  the  cases, 
treated  in  Paris  are  published  quarterly  in  the  Annales  de  rinstitut  Pasteur,  and 
general  summaries  of  the  results  of  each  year  are  also  prepared.  As  we  have  said, 
the  ordinary  mortality  formerly  was  16  per  cent  of  all  persons  bitten.  During  the 
ten  years  1886-95,  Z7»337  cases  were  treated,  with  a  mortality  of  .48  per  cent.  It 
has  been  alleged  that  many  people  are  treated  who  have  been  bitten  by  dogs  that 
were  not  mad.  This,  however,  is  not  more  true  of  the  cases  treated  by  Pasteur's 
method  than  it  was  of  those  on  which  the  ordinary  mortality  of  16  per  cent  was 
based,  and  care  is  taken  in  making  up  the  statistics  to  divide  the  cases  into  three 
classes.  Class  A  includes  only  persons  bitten  by  dogs  proved  to  have  had  rabies,  by 
inoculation  in  healthy  animals  of  parts  of  the  central  nervous  system  of  the  diseased 
animal.  Class  B  includes  those  bitten  by  dogs  that  a  competent  veterinary  surgeon 
has  pronounced  to  be  mad.  Class  C  includes  all  other  cases.  During  1895,  I22  cases 
belonging  to  Class  A  were  treated,  with  no  deaths  ;  949  belonging  to  Class  B,  with 
two  deaths  ;  and  449  belonging  to  Class  C,  with  no  deaths.  Besides  the  Institute  in 
Paris,  similar  institutions  exist  in  other  parts  of  France,  in  Italy,  and  especially  in 
Russia,  as  well  as  in  other  parts  of  the  world  ;  and  in  these  similar  success  has  been 
experienced.  It  may  be  now  taken  as  established,  that  a  very  grave  responsibility  rests 
on  those  concerned,  if  a  person  bitten  by  a  mad  animal  is  not  subjected  to  the  Pasteur 
treatment. 

Antirabic  Serum.  —  In  the  early  part  of  the  nineteenth  century  an 
Italian  physician,  Valli,  showed  that  immunity  against  rabies  could  be 
conferred  by  administering  through  the  stomach  progressively  increasing 
doses  of  hydrophobic  virus.  Following  up  this  observation,  Tizzoni  and 
Centanni  have  attenuated  rabic  virus  by  submitting  it  to  peptic  digestion, 
and  have  immunised  animals  by  injecting  gradually  increasing  strengths 
of  such  virus.  This  method  is  usually  referred  to  as  the  Italian  method 
of  immunisation.  The  latter  workers  showed  from  this  that  the  serum 
of  animals  thus  immunised  could  give  rise  to  passive  immunity  in  other 
animals  ;  and  further,  that  if  injected  into  animals  from  seven  to  fourteen 
days  after  infection  with  the  virus,  it  prevented  the  latter  from  producing 
its  fatal  effects,  even  when  symptoms  had  begun  to  manifest  themselves. 
They  further  succeeded  in  producing  in  the  sheep  and  the  dog  an 
immunity  equal  to  from  1-25,000  to  1-50,000  (vide  p.  472),  and  they 
recommended  the  use,  in  severe  cases,  of  the  serum  of  such  animals  in 
addition  to  the  treatment  of  the  patient  by  the  Pasteur  method.  We  do 
not,  of  course,  know  whether  the  serum  contains  antitoxic  or  antimi- 
crobic  bodies. 

Methods,  (a)  Diagnosis.  —  When  a  person  is  bitten  by  an  animal 
suspected  to  be  rabid,  the  latter  must  under  no  circumstances  be  killed. 
Much  more  can  be  learned  by  watching  it  while  alive  than  by  post- 
mortem examination.  In  the  latter  case  only  such  things  as  the  occur- 
rence of  broken  teeth,  marked  congestion  of  the  fauces;  or  the  presence 
of  unwonted  material  in  the  stomach  throw  any  light  on  the  condition. 
By  examination  of  the  spinal  ganglia  (vide  supra},  an  early  and  pro- 


HYDROPHOBIA. 

visional  diagnosis  may  be  readily  made  which  will  be  confirmed  or  dis- 
missed by  the  results  of  the  slower  but  more  certain  inoculation  method. 
On  the  other  hand,  in  the  living  animal  the  development  of  the  charac- 
teristic symptoms  can  be  watched,  and  death  will  occur  in  not  more  than 
five  days.  If  the  suspected  animal  has  been  killed,  then  a  small  piece 
of  its  medulla  or  cord  must  be  taken,  with  all  aseptic  precautions,  rubbed 
up  in  a  little  sterile  .75  per  cent  sodium  chloride  solution,  and  injected 
by  means  of  a  syringe  beneath  the  dura  mater  of  a  rabbit,  the  latter 
having  been  trephined  over  the  cerebrum  by  means  of  the  small  trephine 
which  is  made  for  the  purpose.  Symptoms  usually  occur  in  from  twelve 
to  twenty-three  days  and  death  in  fifteen  to  twenty-five  days.  When 
such  inoculation  has  to  be  practised  it  is  evident  that  the  diagnosis  is 
delayed.  When  the  material  for  inoculation  has  to  be  sent  any  distance 
this  is  best  effected  by  packing  the  head  of  the  animal  in  ice.  The 
virulence  of  organs  is  not  lost,  however,  if  they  are  simply  placed  in 
sterile  water  or  glycerin  in  well-stoppered  bottles. 

(b)  Treatment.  —  Every  wound  inflicted  by  a  rabid  animal  ought  to 
be  cauterised  with  the  actual  cautery  as  soon  as  possible.  By  such  treat- 
ment the  incubation  period  will  at  any  rate  be  lengthened,  and  therefore 
there  will  be  better  opportunity  for  the  Pasteur  inoculation  method  being 
efficacious.  The  person  ought  then  to  be  sent  to  the  nearest  Pasteur 
Institute  for  treatment.  It  is  of  great  importance  that  in  such  a  case 
the  nervous  system  of  the  animal  should  also  be  sent,  in  order  that  the 
diagnosis  may  be  certainly  verified. 


APPENDIX   C. 
MALARIAL  FEVER. 

IT  has  now  been  conclusively  proved  that  the  cause  of  malarial  fever 
is  a  protozoon  of  which  there  are  several  species.  The  parasite  was 
formerly  known  as  the  h&matozoon  or  plasmodium  malaria,  although 
the  use  of  the  latter  term  is  incorrect ;  the  term  hcemamceba  is,  however, 
now  generally  employed.  The  parasite  was  first  observed  by  Laveran 
in  1880,  and  his  discovery  received  confirmation  from  the  independent 
researches  of  Marchiafava  and  Celli,  and  later  from  the  researches  of 
many  others  in  various  parts  of  the  world.  Golgi  supplied  valuable 
additional  information,  especially  in  relation  to  the  sporulation  of  the 
organism  and  the  varieties  in  different  types  of  malarial  fever.  In  this 
country  valuable  work  on  the  subject  was  done  by  Manson,  and  to  him 
specially  belongs  the  credit  of  regarding  the  exflagellation  of  the 
organism  as  a  preparation  for  an  extra-corporeal  phase  of  existence. 
By  induction  he  arrived  at  the  belief  that  the  cycle  of  existence  out- 
side the  human  body  probably  took  place  in  the  mosquito.  It  was 
specially  in  order  to  discover,  if  possible,  the  parasite  in  the  mosquito, 
that  Ross  commenced  his  long  series  of  observations,  which  were  ulti- 
mately crowned  with  success.  After  patient  and  persistent  search,  he 
found  rounded  pigmented  bodies  in  the  wall  of  the  stomach  of  a  dapple- 
winged  mosquito  (a  species  of  Anopheles)  which  had  been  fed  on  the 
blood  of  a  malarial  patient.  The  pigment  in  these  bodies  was  exactly 
similar  to  that  in  the  malarial  parasite,  and  he  excluded  the  possibility 
of  their  representing  anything  else  than  a  stage  in  the  life  cycle  of  the 
organism.  He  confirmed  this  discovery  and  obtained  corresponding 
results  in  the  case  of  the  proteosoma  infection  of  birds,  where  the  para- 
site is  closely  related  to  that  of  malaria.  In  birds  affected  with  this 
organism,  he  was  able  to  trace  all  stages  of  its  development,  from  the 
time  it  entered  the  stomach  along  with  the  blood,  till  the  time  when  it 
settled  in  a  special  form  in  the  salivary  glands  of  the  insect.  Ross's 
results  were  published  in  1898.  Exactly  corresponding  stages  were 
afterwards  found  in  the  case  of  the  different  species  of  the  human  para- 
site, by  Grassi,  Bignami,  and  Bastianelli ;  and  these  and  other  Italian 
observers  also  supplied  important  information  regarding  the  transmission 


MALARIAL   FEVER. 

of  the  disease  by  infected  mosquitoes.  Abundant  additional  observa- 
tions, with  confirmatory  results,  were  supplied  by  Koch,  Daniels,  Chris- 
tophers, Stephens,  and  others.  Wherever  malaria  has  been  studied  the 
result  has  been  the  same.  Lastly,  we  may  mention  the  striking  experi- 
ment carried  out  by  Manson  by  means  of  mosquitoes  fed  on  the  blood 
of  patients  in  Italy  suffering  from  mild  tertian  fever.  The  insects,  after 
being  thus  fed,  were  taken  to  London  and  allowed  to  bite  the  human 
subject,  Hanson's  son,  Dr.  P.  Thurburn  Manson,  offering  himself  for 
the  purpose.  The  result  was  that  infection  occurred;  the  parasites 
appeared  in  the  blood,  and  were  associated  with  an  attack  of  tertian 
fever.  Ross's  discovery  has  not  only  been  a  means  of  elucidating  the 
mode  of  infection,  but,  as  will  be  shown  below,  has  also  supplied  the 
means  of  successfully  combating  the  disease. 

From  the  zoological  point  of  view  the  mosquito  is  regarded  as  the 
definitive  host  of  the  parasite,  the  human  subject  as  the  intermediate 
host.  But  in  describing  the  life  history,  it  will  be  convenient  to  con- 
sider, first,  the  cycle  in  the  human  body,  and,  secondly,  that  in  the 
mosquito. 

The  Cycle  in  the  Human  Subject.  —  With  regard  to  this  cycle,  it 
may  be  stated  that  the  parasite  is  conveyed  by  the  bite  of  the  mosquito 
in  the  form  of  a  small  filamentous  blast  or  exotospore,  which  gives  rise 
to  the  small  amoeboid  organism  or  ameebula  seen  in  the  human  blood. 
There  is  then  a  regularly  repeated  asexual  cycle  of  the  parasite  in  the 
blood,  which  cycle  determines  the  type  of  the  fever,  e.g.  the  cycle  of 
the  tertian  parasite  is  completed  in  forty-eight  hours,  although  a  double 
infection  with  this  parasite  may  produce  a  quotidian  type.  During  this 
cycle  there  is  a  growth  of  the  amcebulae  within  the  red  corpuscles  up  to 
their  complete  development  and  sporulation.  The  onset  of  the  febrile 
attack  corresponds  with  the  stage  of  sporulation  and  the  -setting  free  of 
the  spores,  or  youngest  forms,  i.e.  with  the  production  of  a  fresh  brood 
of  parasites.  These  spores  soon  become  attached  to,  and  penetrate 
into  the  interior  of,  the  red  corpuscles,  becoming  intra-corpuscular 
amoebulae ;  the  cycle  is  thus  completed.  The  parasites  are  most 
numerous  in  the  blood  during  the  development  of  the  pyrexia,  and, 
farther,  they  are  also  much  more  abundant  in  the  internal  organs  than 
in  the  peripheral  blood ;  in  the  malignant  type,  for  example,  the  process 
of  sporulation  is  practically  confined  to  the  former. 

In  addition  to  these  forms,  which  are  part  of  the  ordinary  cycle, 
there  are  derived  from  the  amoebulae  other  forms,  which  are  called 
gametes,  or  sexual  cells.  These  gametes  remain  unaltered  during  suc- 
cessive attacks  of  pyrexia,  and  undergo  no  further  change  until  the 
blood  is  removed  from  the  human  body.  In  the  simple  tertian  and 
quartan  fevers  (vide  infra]  the  gametes  closely  resemble  in  structure 


5i8 


APPENDIX   C. 


the  fully  developed  amoebulae  before  sporulation,  whereas  in  the  malig- 
nant type  they  have  a  characteristic  crescent-like  or  sausage-shaped 
form ;  hence  they  are  often  spoken  of  as  "  crescentic  bodies." 


FIG.  157. 


FIG.  158. 


FIG.  159. 


FIG.  160. 


• 


o 

4 


FIG.  161.  FIG.  162. 

FIGS.  157-162.  —  Various  phases  of  the  benign  tertian  parasite. 

Fig.  157.  Several  young  ring-shaped  amoebulae  within  the  red  corpuscles,  one  of  the  latter  en- 
larged and  showing  a  dotted  appearance.  Fig.  158.  A  larger  amcebula  containing  pigment  granules. 
Fig.  159.  Two  large  amoebulae,  exemplifying  the  great  variation  in  form.  Fig.  160.  Large  amcebulse 
assuming  the  spherical  form  and  showing  isolated  fragments  of  chromatin  —  preparatory  to  sporulation. 
Fig.  161.  Sporocyte,  which  has  produced  eighteen  spores,  each  of  which  contains  a  small  collection  of 
chromatin.  Fig.  162.  A  number  of  spores  which  have  just  been  set  free  in  the  plasma,  x  1000. 

The  various  forms  of  the  parasite  seen  in  the  human  blood  may  now 
be  described  more  in  detail. 


MALARIAL   FEVER. 


519 


i.  The  Spores  or  Enh&mospores  (Lankester)  are  the  youngest  and 
smallest  forms  resulting  from  the  segmentation  of  the  adult  atnoabula  or 
sporocyte.  They  are  of  round  or  oval  shape  and  of  small  size  usually 


FIG.  163. 


FIG.  164. 


FIG.  165. 


>;'"?• 
FIG.  166. 


kW    ^ 

FIG.  167.  FIG.  168. 

FIGS.  163-168.  —  Exemplifying  phases  of  the  malignant  parasite. 

Fig.  163.  Two  small  ring-shaped  amrebulae  within  the  red  corpuscles.  Fig.  164.  A  "crescent" 
or  gamete  showing  the  envelope  of  the  red  corpuscles;  also  an  amoebula.  Figs.  165-168  illustrate  the 
changes  in  form  undergone  by  the  crescents  outside  the  body.  In  the  interior  of  the  spherical  form  in 
Fig.  167  evidence  of  the  flagella  can  be  seen.  Fig.  168.  A  male  gamete  which  has  undergone  exflagella- 
tion,  showing  the  thread-like  microgametocytes  or  spermatozoa  attached  at  the  periphery,  x  1000. 
(The  figures  in  this  plate  are  from  preparations  kindly  lent  by  Dr.  Manson.) 

not  exceeding  2  /x  in  diameter ;  the  size,  however,  varies  somewhat  in 
the  different  types  of  fever.     A  nucleus  and  peripheral  protoplasm  can 


520  APPENDIX    C. 

be  distinguished  (Figs.  161,  162).  The  former  appears  as  a  small 
rounded  body  which  usually  remains  unstained,  but  contains  a  minute 
mass  of  chromatin  which  stains  a  deep  red  with  the  Romanowsky 
method  ;  the  peripheral  protoplasm  is  coloured  fairly  deeply  with  methy- 
lene-blue.  The  spores  show  little  or  no  amoeboid  movement ;  at  first 
free  in  the  plasma,  they  soon  attack  the  red  corpuscles,  where  they  be- 
come the  intra-corptiscular  amoebulae.  If  the  blood,  say  in  a  mild  ter- 
tian case,  be  examined  in  the  early  stages  of  pyrexia,  one  often  finds  at 
the  same  time  sporulating  forms,  free  spores,  and  the  young  amoebulae 
within  the  red  corpuscles. 

2.  Infra-corpuscular  Bodies  or  Am&bulce. —  These  include  the  para- 
sites which  have  attacked  the  red  corpuscles ;  they  are  at  first  situated 
on  the  surface  of  the  latter,  but  afterwards  penetrate  their  substance. 
They  usually  occur  singly  in  the  red  corpuscles,  but  sometimes  two  or 
more  may  be  present  together.  The  youngest  or  smallest  forms  appear 
as  minute  colourless  specks,  of  about  the  same  size  as  the  spores.  As 
seen  in  fresh  blood,  they  exhibit  more  or  less  active  amoeboid  move- 
ment, showing  marked  variations  in  shape.  The  amount  and  character 
of  the  amoeboid  movement  varies  somewhat  in  different  types  of  fever. 
As  they  increase  in  size,  pigment  appears  in  their  interior  as  minute 
dark-brown  or  black  specks,  and  gradually  becomes  more  abundant 
(Figs.  158,  159).  The  pigment  may  be  scattered  through  their  sub- 
stance, or  concentrated  at  one  or  more  points,  and  often  shows  vibratory 
or  oscillating  movements.  This  pigment  is  no  doubt  elaborated  from 
the  haemoglobin  of  the  red  corpuscles,  the  parasite  growing  at  the  ex- 
pense of  the  latter.  The  red  corpuscles  thus  invaded  may  remain  unal- 
tered in  appearance  (quartan  fever),  may  become  swollen  and  pale 
(tertian  fever),  or  somewhat  shrivelled  and  of  darker  tint  (malignant 
fever).  In  stained  specimens  a  nucleus  may  be  seen  in  the  parasite  as 
a  pale  spot  containing  chromatin  which  may  be  arranged  as  a  single 
concentrated  mass  or  as  several  separated  granules,  the  chromatin  being 
coloured  a  deep  red  by  the  Romanowsky  method.  The  protoplasm  of 
the  parasite,  which  is  coloured  of  varying  depth  of  tint  with  methylene- 
blue,  shows  great  variation  in  configuration  (Fig.  159).  The  young 
parasites  not  unfrequently  present  a  "  ring-form,"  a  portion  of  the  red 
corpuscle  being  thus  enclosed  by  the  parasite.  These  ring-forms  are 
met  with  in  all  the  varieties  of  the  parasite,  but  they  are  especially  com- 
mon in  the  case  of  the  malignant  parasite,  where  they  are  of  smaller 
size  and  of  more  symmetrical  form  than  in  the  others,  representing  a 
quiescent  stage  (Fig.  163)  ;  the  pigment  is  usually  collected  in  a  small 
clump  at  one  side. 

Within  the  red  corpuscles  the  parasites  gradually  increase  in  size  till 
the  full  adult  form  is  reached  (Fig.  160).  In  the  latter  stage  the  para- 


MALARIAL   FEVER. 


521 


site  loses  its  amoeboid  movement  more  or  less  completely,  has  a  some- 
what rounded  form,  and  contains  a  considerable  amount  of  pigment. 
In  the  malignant  form  it  only  occupies  a  fraction  of  the  red  corpuscle. 
The  adult  parasites  may  then  undergo  sporulation,  but  not  all  of  them 
do  so ;  some  become  degenerated  and  ultimately  break  down. 

3.  Sporocytes  or  Sporulation  Forms.  —  In  the  largest  amoebulse 
before  sporulation  the  nuclear  chromatin  becomes  scattered  throughout 
the  parasite.  During  sporulation  the  pigment  becomes  collected  as  a 
more  or  less  central  mass,  and  the  protoplasm  segments  into  a  number 
of  spores,  each  of  which  contains  a  small  mass  of  chromatin  (Fig.  161). 
The  spores  are  of  rounded  or  oval  shape,  as  above  described,  and  are 
set  free  by  the  rupture  of  the  envelope  of  the  red  corpuscle.  The  pig- 
ment also  becomes  free  and  may  be  taken  up  by  leucocytes.  The  num- 
ber and  arrangement  of  the  spores  within  the  sporocyte  varies  in  the 
different  types.  In  the  quartan  there  are  6-12,  and  the  segmentation  is 
in  a  radiate  manner,  giving  rise  to  the  characteristic  daisy-head  appear- 
ance;  in  the  tertian  they  number  15-20,  and  have  a  somewhat  rosette- 
like  arrangement  (Fig.  161)  \  in  the  malignant  there  are  usually  6-12 
spores  of  small  size  and  somewhat  irregularly  arranged. 

Gametes. — As  stated  above,  these  are  sexual  cells  which  are  formed 
from  certain  of  the  amoebulse,  and  which  undergo  no  further  develop- 
ment in  the  human  subject.  In  the  mild  tertian  and  quartan  fevers  they 
resemble  somewhat  the  largest  amoebulse,  the  female  cells  being  rather 
more  granular  in  appearance  than  the  male.  In  the  malignant  fevers 
the  gametes  have  the  special  crescentic  form  mentioned  above.  They 
measure  8-9  ^  in  length,  arid  occasionally  a  fine  curved  line  is  seen  join- 
ing the  extremities  on  the  concave  aspect,  which  represents  the  envelope 
of  the  red  corpuscle  (Fig.  164).  They  are  colourless  and  transparent, 
and  are  enclosed  by  a  distinct  membrane ;  in  the  central  part  there  is 
a  collection  of  pigment  and  granules  of  chromatin.  It  is  stated  that  the 
male  crescents  can  be  distinguished  from  the  female  by  their  appearance. 
In  the  former  the  pigment  is  less  dark  and  more  scattered  than  in  the 
latter,  and  there  are  several  granules  of  chromatin ;  in  the  latter  the 
pigment  is  dark  and  concentrated,  often  in  a  small  ring,  and  there  are 
one  or  two  masses  of  chromatin  in  the  centre  of  the  crescent.  Accord- 
ing to  the  Italian  observers  the  early  forms  of  the  crescents  are  some- 
what fusiform  in  shape  and  are  produced  in  the  bone-marrow.  The  fully 
developed  crescents  do  not  appear  in  the  blood  till  several  days  after 
the  onset  of  the  fever,  and  they  may  be  found  a  considerable  time  after 
the  disappearance  of  the  pyrexial  attacks.  They  are  also  little,  if  at  all, 
influenced  by  the  administration  of  quinine. 

The  Cycle  in  the  Mosquito.  —  As  already  explained,  this  starts  from 
the  gametes.  After  the  blood  is  shed,  or  after  it  is  swallowed  by  the  mos- 


522  APPENDIX   C. 

quito,  two  important  phenomena  occur,  viz.,  (a)  the  exflagellation  of  the 
male  gamete,  and  (b)  the  impregnation  of  the  female.  If  the  blood  from 
a  case  of  malignant  infection  be  examined  in  a  moist  chamber,  preferably 
on  a  warm  stage,  under  the  microscope,  both  male  and  female  gametes 
may  be  seen  to  become  oval  and  afterwards  rounded  in  shape  (Figs. 
165-167).  Thereafter,  in  the  case  of  the  male  cell,  a  vibratile  or  danc- 
ing movement  of  the  pigment  granules  can  be  seen  in  the  interior,  and 
soon  several  flagella-like  structures  shoot  out  from  the  periphery  (Fig. 
1 68).  They  are  of  considerable  length  but  of  great  fineness,  and  often 
show  a  somewhat  bulbous  extremity.  By  the  Romanowsky  method  they 
have  been  found  to  contain  a  delicate  core  of  chromatin,  which  is  covered 
by  protoplasm.  They  represent  the  male  cells  proper,  that  is,  they  are 
sperm-cells  or  spermatozoa ;  they  are  also  known  as  microgametocytes. 
They  become  detached  from  the  sphere  and  move  away  in  the  surround- 
ing fluid.  One  of  them  may  enter  a  female  gamete  and  thus  a  process 
of  true  impregnation  occurs.  It  is  also  stated  that  the  female  cell 
before  fertilisation  gives  off  two  polar  bodies.  This  was  first  observed  by 
MacCallum  in  the  case  of  halteridium,  and  he  found  that  the  female  cell 
afterwards  acquired  the  power  of  independent  movement  or  became  a 
"  travelling  vermicule."  He  also  observed  the  impregnation  of  the  malig- 
nant parasite.  The  fertilised  female  cell  is  now  generally  spoken  of  as 
a  zygote. 

It  has  been  established  that  the  phenomena  just  described  occur 
within  the  stomach  of  the  mosquito,  and  that  the  fertilised  cell  or  zygote 
penetrates  the  stomach  wall  and  settles  between  the  muscle  fibres ;  on 
the  second  day  after  the  mosquito  has  ingested  the  infected  blood  small 
rounded  cells  about  6-8  /x  in  diameter  and  containing  clumps  of  pigment 
may  be  found  in  this  position.  (It  was  in  fact  the  character  of  the  pig- 
ment which  led  Ross  to  believe  that  he  had  before  him  a  stage  in  the 
development  of  the  malarial  parasite.)  A  distinct  membrane  called 
a  sporocyst  forms  around  the  zygote,  and  on  subsequent  days  a  great 
increase  in  size  takes  place,  and  the  cysts  come  to  project  from  the  sur- 
face of  the  stomach  into  the  body  cavity.  The  zygote  divides  into  a 
number  of  cells  called  blastophores  or  spore-mother-cells,  and  on  the  sur- 
face of  each  there  are  formed  a  large  number  of  filiform  spores  which 
have  a  radiate  arrangement ;  these  were  called  by  Ross  "  germinal  rods," 
but  are  now  usually  known  as  zygotoblasts  or  exotospores  (in  contradistinc- 
tion to  the  enhsemospores  of  the  human  cycle) .  The  full  development 
within  the  sporocyst  occupies,  in  the  case  of  proteosoma,  about  seven 
days,  in  the  case  of  the  malarial  parasites  a  little  longer.  When  fully 
developed  the  cyst  measures  about  60  p.  in  diameter,  and  appears  packed 
with  zygotoblasts.  It  then  bursts,  and  the  latter  are  set  free  in  the  body 


f      V  OF  THE 

fl    UNIVERSITY 

MALARIAL   FEVER. 


cavity.  A  large  number  settle  within  the  large  veneno-salivary  gland  of 
the  insect,  and  are  thus  in  a  position  to  be  injected  along  with  its  secre- 
tion into  the  human  subject.  Daniels  found  that  in  the  case  of  the 
malignant  parasite  an  interval  of  twelve  days  at  least  intervened  between 
the  time  of  feeding  the  mosquito  and  the  appearance  of  the  filiform 
spores  in  the  gland. 

It  will  thus  be  seen  that  in  the  human  subject  the  parasite  passes 
through  an  indefinite  number  of  regularly  recurring  asexual  cycles,  with 
the  giving  off  of  collateral  sexual  cells,  and  that  in  the  mosquito  there  is 
one  cycle  which  may  be  said  to  start  with  the  impregnation  of  the  female 
gametocyte  —  a  cycle,  moreover,  in  which  the  parasite  reaches  the  most 
advanced  stage  of  its  development. 

Varieties  of  the  Malarial  Parasite.  —  The  view  propounded  by  La- 
veran  was  that  there  is  only  one  species  of  malarial  parasite,  which  is  poly- 
morphous, and  presents  slight  differences  in  structural  character  in  the 
different  types  of  fever.  It  may,  however,  now  be  accepted  as  proved 
that  there  are  at  least  three  distinct  species  which  infect  the  human  sub- 
ject. This  is  shown  by  their  distinct  morphological  characters,  by  differ- 
ences in  the  length  of  their  cycle  of  development,  and  also,  to  a  certain 
extent,  by  their  pathogenic  effects.  Practically  all  are  agreed  as  to  a 
•division  into  two  groups,  one  of  which  embraces  the  parasites  of  the 
milder  fevers,  —  "winter-spring"  fevers  of  Italian  writers,  —  there  being 
here  two  distinct  species,  for  the  quartan  and  tertian  types  respectively  ; 
whilst  the  other  includes  the  parasites  of  the  severer  forms  —  "  aestivo- 
-autumnal  "  fevers,  malignant  or  pernicious  fevers  of  the  tropics,  or  irregu- 
larly remittent  fevers.  There  is  still  doubt  as  to  whether  there  are  more 
than  one  species  in  this  latter  group.  Formerly,  Italian  writers  distin- 
guished (i)  a  quotidian,  (2)  a  non-pigmented  quotidian,  and  (3)  a 
malignant  tertian  parasite,  though  the  morphological  differences  de- 
scribed were  slight.  Further  observations  have,  however,  thrown  doubt 
on  this  distinction,  and  the  evidence  rather  goes  to  show  that  there  is  a 
single  species,  which  probably  has  a  cycle  of  forty-eight  hours,  though 
variations  may  occur  ;  multiple  infection  is  moreover  common,  and  thus 
a  quotidian  or  irregular  type  may  occur.  Manson,  for  example,  consid- 
ers that  if  there  exists  a  true  quotidian  fever  it  must  be  of  the  rarest 
occurrence.  Although  the  question  cannot  be  considered  as  finally  set- 
tled, we  shall  accordingly  speak  of  three  species  of  human  parasites. 
The  zoological  position  may  be  shown  by  the  following  scheme,  generally 
followed  by  English  writers,  the  terminology  being  chiefly  that  of  Grassi 
and  Feletti  :  — 


524  APPENDIX   C. 

Family :  H^MAMCEBID^  (Wasielewski) 

Genus  I.  I laemamoeba.  The  mature  gametes  resemble  in  form  the  sporocytes- 
before  they  have  differentiated  into  spores. 

Species  I.     Hccmamoeba  danilewski  or  halteridium* 
Parasite  of  pigeons,  crows,  etc. 

Species  2.     Hcemamceba  relicta  or  proteosoma. 
Parasite  of  sparrows,  larks,  etc. 

Species  3.     ffcemamceba  malaria, 

Parasite  of  quartan  fever  of  man. 

Species  4.     ffesmamceba  vivax. 

Parasite  of  tertian  fever  of  man. 

Genus  II.      Hsemomenas.     The  gametes  have  a  special  crescentic  form. 
Species:  Hamomenas prcecox. 

Parasite  of  malignant  or  sestivo-autumnal  fever  of  man. 

In  addition  there  are  other  species  belonging  to  the  same  family  of 
blood  parasites,  which  infect  frogs,  lizards,  bats,  etc.,  especially  in 
malarial  regions. 

We  shall  now  give  the  chief  distinctive  characters  of  the  three 
human  parasites. 

1.  Parasite  of  Quartan  Fever.  —  The  cycle  of  development  in  man 
is  seventy-two  hours,  and  produces  pyrexia  every  third  day ;  double  or 
triple  infection  may,  however,  occur.     In  fresh  specimens  of  blood  the 
outline  is  more  distinct  than  that  of  the  tertian  parasite,  and  amoeboid 
movement  is  less  marked.     Only  the   smaller  forms   show   movement^ 
and  this  is  not  of  active  character.     The  infected  red  corpuscles  do  not 
become  altered  in  size  or  appearance,  and  the  pigment  within  the  para- 
site is  in  the  form  of  coarse  granules,  of  dark  brown  or  almost  black 
colour.    The  fully  developed  sporocyte  has  a  "  daisy-head  "  appearance, 
'dividing  by  regular  radial  segmentation  into  six  to  twelve  spores,  which, 
on  becoming  free,  are  rounded  in  form. 

2.  The  Parasite  of  Mild  Tertian  Fever.  —  The  cycle  of  development 
is  completed  in  forty-eight  hours,  though  a  quotidian  type  of  fever  may 
be  produced  by  double  infection.    The  amoebulae  have  a  less  refractile 
margin  than  in  the  quartan  type,  and  are  thus  less  easily  distinguished 
in  the  fresh  blood;  the  amoeboid  movements  are,  however,  much  more 
active,  while  longer   and   more  slender  processes  are  given  off.     The 
infected  corpuscles   become   swollen  and   pale,  and  may  show  deeply 
stained  points  by  the  Romanowsky  -method  —  "Schiiffner's  dots."     The 
pigment  within  the  parasite  is  fine  and  of  yellowish-brown  tint.     The 
mature  sporocyte  is  rather  larger  than  in  the  quartan,  has  a   rosette 
appearance,  and  gives  rise  to  fifteen  to  twenty  spores,  which  have  a 
somewhat  oval  shape. 


MALARIAL   FEVER.  525 

In  both  the  quartan  and  tertian  fevers  all  the  stages  of  development 
can  be  readily  observed  in  the  peripheral  blood. 

3 .  The  Parasite  of  Malignant  or  sEstivo-autumnal  Fever,  or  Tropical 
Malaria.  —  The  cycle  in  the  human  subject  probably  occupies  forty- 
eight  hours,  though  this  cannot  be  definitely  stated  to  be  always  the  case 
(vide  supra}.  The  amcebulae  in  the  red  corpuscles  are  of  small  size, 
and  their  amoeboid  movements  are  very  active  ;  they  often,  however, 
pass  into  the  quiescent  ring  form  (Fig.  163).  The  pigment  granules, 
even  in  the  larger  forms,  are  few  in  number  and  very  fine ;  the  infected 
red  corpuscles  have  a  tendency  to  shrivel  and  assume  a  deeper  or  coppery 
tint.  The  fully  developed  sporocyte  occupies  less  than  half  the  red 
corpuscle,  and  gives  rise  to  usually  from  six  to  twelve  spores,  somewhat 
irregularly  arranged  and  of  minute  size.  Sporulation  takes  place  almost 
exclusively  in  the  internal  organs,  spleen,  etc.,  so  that,  as  a  rule,  no  sporo- 
cytes  can  be  found  in  the  blood  taken  in  the  usual  way.  The  proportion 
of  red  corpuscles  infected  by  the  amcebulae  is  also  much  larger  in  the  inter- 
nal organs.  The  gametes  have  the  crescentic  form,  as  already  described. 

Cases  of  infection  with  the  malignant  parasite  sometimes  assume  a 
pernicious  character,  and  then  the  number  of  organisms  in  the  interior 
of  the  body  may  be  enormous.  In  certain  fatal  cases  with  coma  the 
cerebral  capillaries  appear  to  be  almost  filled  with  them,  many  being  in 
process  of  sporulation,  and  in  so-called  algid  cases,  characterised  by  great 
collapse,  a  similar  condition  has  been  found  in  the  capillaries  of  the 
omentum  and  intestines.  The  process  of  blood  destruction  present  in 
all  malarial  fevers,  reaches  its  maximum  in  the  malignant  class,  and  the 
brown  or  black  pigment  elaborated  by  the  parasites  —  in  part  after  being 
taken  up  by  leucocytes,  chiefly  of  the  mononuclear  class  —  becomes 
deposited  in  various  organs,  spleen,  liver,  brain,  etc.,  especially  in  the 
endothelium  of  vessels  and  the  perivascular  lymphatics.  In  the  severer 
forms  also  brownish-yellow  pigment  is  apparently  derived  from  liberated 
haemoglobin,  and  accumulates  in  various  parts,  especially  in  the  liver 
cells ;  most  of  this  latter  gives  the  reaction  of  an  iron  salt. 

General  Considerations.  —  The  developments  of  the  malarial  parasites 
in  the  mosquito  and  infection  of  the  human  subject  through  the  bites  of 
this  insect  have,  by  the  work  of  Ross  and  others,  as  detailed  above, 
become  established  scientific  facts.  These  facts,  moreover,  point  to 
certain  definite  methods  of  prevention  of  infection,  and  these  methods 
have  to  a  certain  extent  already  been  practically  tested.  The  extensive 
observations  recently  carried  out  go  to  show  that  all  the  mosquitoes 
which  act  as  hosts  of  the  parasite  belong  to  the  genus  anopheles;  of 
these  there  are  a  large  number  of  species,  and  in  at  least  eight  or  nine 
the  parasite  has  been  found.  The  breeding  places  of  these  insects  are 
chiefly  in  stagnant  pools  and  other  collections  of  standing  water,  and 


526  APPENDIX   C. 

accordingly  the  removal,  where  practicable,  by  drainage  of  such  col- 
lections in  the  vicinity  of  centres  of  population,  and  the  killing  of  the 
larvae  by  petroleum  sprinkled  on  the  water,  have  constituted  one  of 
the  most  important  measures.  This  has  been  carried  out  recently 
at  Freetown  by  Logan  Taylor,  under  the  superintendence  of  Ross,  and 
the  result  has  been  to  show  that  a  marked  lowering  of  the  number  of 
cases  may  be  effected  at  comparatively  small  cost.  Another  measure  is 
the  protection  against  mosquito  bites  by  netting,  it  being  fortunately  the 
habit  of  the  anopheles  to  rarely  become  active  before  sundown.  The 
experiments  of  Sambon  and  Low  in  the  Campagna  have  proved  that 
individuals  using  these  means  of  protection  may  live  in  a  highly  malarial 
district  without  becoming  infected.  The  administration  of  quinine  to 
those  in  highly  malarial  regions,  in  order  to  prevent  infection,  has  also 
been  recommended  and  carried  out.  In  the  tropics  the  natives  in  large 
proportion  suffer  from  malarial  infection,  and  one  would  accordingly  ex- 
pect that  infection  of  the  mosquitoes  in  the  neighbourhood  of  native  settle- 
ments will  be  common.  This  has  been  found  to  be  actually  the  case,  and 
it  has  accordingly  been  suggested  that  the  dwellings  of  whites  should  as 
far  as  possible  be  at  some  distance  from  the  native  centres  of  population. 

So  far  none  of  the  lower  animals  have  been  found  to  act  as  inter- 
mediate hosts  to  the  parasite  of  human  malaria,  but  the  possibility  of 
such  being  the  case  cannot  be  as  yet  definitely  excluded.  On  the  death 
of  infected  mosquitoes  the  exotospores  or  sporozoites  will  become  set  free, 
and  therefore  theoretically  there  is  a  possibility  that  they  may  enter  the 
human  subject  by  inhalation  or  by  some  other  means.  We  have  no 
facts,  however,  to  show  that  this  really  occurs,  and  the  evidence  already 
obtained  establishes  the  bites  of  mosquitoes  as  the  most  important  if  not 
the  only  mode  of  infection. 

It  may  also  be  mentioned  as  a  scientific  fact  of  some  interest,  though 
not  bearing  on  the  natural  modes  of  infection,  that  the  disease  can  also 
be  communicated  from  one  person  to  another  by  injecting  the  blood 
containing  the  parasites.  Several  experiments  of  this  kind  have  been 
performed  (usually  about  \  to  i  c.c.  of  blood  has  been  used),  and  the 
result  is  more  certain  in  intravenous  than  in  subcutaneous  injection.  In 
such  cases  there  is  an  incubation  period,  usually  of  from  seven  to  four- 
teen days,  after  which  the  fever  occurs.  The  bulk  of  evidence  goes  to 
show  that  the  same  type  of  fever  is  reproduced  as  was  present  in  the 
patient  from  whom  the  blood  was  taken. 

Methods  of  Examination.  —  The  parasites  may  be  studied  by  examin- 
ing the  blood  in  the  fresh  condition,  or  by  permanent  preparations. 
In  the  former  case,  a  slide  and  cover-glass  having  been  thoroughly 
cleaned,  a  small  drop  of  blood  from  the  finger  or  lobe  of  the  ear  is  caught 
by  the  cover-glass,  and  allowed  to  spread  out  between  it  and  the  slide. 


MALARIAL   FEVER.  527 

It  ought  to  be  of  such  a  size  that  only  a  thin  layer  is  formed.  A  ring  of 
vaseline  is  placed  round  the  edge  of  the  cover-glass  to  prevent  evapora- 
tion. For  satisfactory  examination  an  immersion  lens  is  to  be  preferred. 
The  amreboid  movements  are  visible  at  the  ordinary  room  temperature, 
though  they  are  more  active  on  a  warm  stage.  With  an  Abbe  condenser 
a  small  aperture  of  the  diaphragm  should  be  used. 

Permanent  preparations  are  best  made  by  means  of  dried  films.  A 
small  drop  of  blood  is  allowed  to  spread  itself  out  between  two  cover- 
glasses,  which  are  separated  by  sliding  the  one  on  the  other.  The  films 
are  then  allowed  to  dry.  A  very  good  method  is  that  of  Manson,  who 
catches  the  drop  of  blood  on  a  piece  of  gutta-percha  tissue  (a  piece  of 
cigarette-paper  also  does  well),  and  then  makes  a  film  on  a  clean  slide 
by  drying  the  blood  over  the  surface.  The  dried  films  are  then  fixed  by 
one  of  the  methods  already  given  (p.  90),  or  by  placing  in  absolute 
alcohol  for  five  minutes  (Manson).  The  films  thus  prepared  and  fixed 
may  be  stained  for  two  or  three  minutes  in  a  saturated  watery  solution 
of  methylene-blue  or  in  carbol-thionin-blue  (p.  101)  ;  the  solutions  must 
be  carefully  filtered  (especially  the  latter),  and  the  films  must  be  washed 
well  after  staining.  They  are  then  dried  and  mounted  in  balsam.  In 
the  case  of  thionin-blue,  sharper  results  are  obtained  by  dehydrating  in 
alcohol  and  clearing  in  xylol  before  mounting.  A  double  stain  may  be 
obtained  by  staining  first  with  .5  per  cent  solution  of  "  alcohol-soluble  " 
eosin  in  methylated  spirit  for  two  minutes,  then  washing  in  water  and 
drying  ;  thereafter  the  blue  stain  is  used  as  above  ;  the  blood  corpuscles 
are  stained  red,  the  parasites  and  nuclei  of  the  leucocytes  blue. 

The  structure  of  the  parasites  is  specially  well  brought  out  by  the 
following  method  of  Rd.  Muir.  The  films  are  fixed  in  saturated  solution 
of  corrosive  sublimate  for  a  few  seconds,  and  are  then  washed  well  in 
running  water.  They  are  then  stained  with  haemalum  for  ten  minutes, 
washed  well,  and  again  stained  for  about,  the  same  time  in  a  saturated 
watery  solution  of  methylene-blue;  they  are  then  washed  in  water, 
dehydrated,  cleared  in  xylol,  and  mounted  in  balsam.  Here  also  eosin 
may  first  be  used  as  a  contrast  stain  ;  but  the  method  as  just  given  is 
-specially  good  for  picking  out  the  parasites  in  the  blood.  The  chromatin 
of  the  parasites  is  coloured  a  violet-blue,  the  protoplasm  a  pure  blue. 

Romanowsky  Method.  —  For  studying  details  in  the  structure,  this 
method  has  been  extensively  applied.  It  depends  on  the  principle  that 
when  "  ripened  "  methylene-blue  is  mixed  with  eosin  a  new  compound  is 
formed  which  has  a  special  affinity  for  the  chromatin  of  the  malarial  para- 
site, staining  it  a  bright  red.  It  is  to  be  noted,  however,  that  the  method 
only  succeeds  with  certain  kinds  of  methylene-blue  and  eosin.  There 
are  various  modes  of  making  and  applying  the  stain  :  we  give  two  recom- 
mended by  Leishman. 


528  APPENDIX    C. 

Solutions  :  A.  Medicinal  methylene-blue  (Griibler)  in  i  per  cent 
watery  solution,  with  .5  per  cent  sodium  carbonate  added.  The  solution 
is  heated  for  about  twelve  hours  at  65°  C.,  and  then  kept  for  about  a 
week  at  warm  room  temperature. 

B.  Eosin  "extra  B.A."  (Griibler)  in  1:1000  watery  solution. 

First  Method.  —  Dilute  some  of  A  and  B,  each  with  25  vols.  of 
water.  Then  mix  equal  parts  (say  2  c.c.)  of  the  diluted  stains.  Stain 
films  (fixed  in  alcohol  and  ether  or  in  alcohol)  for  half  an  hour  or  longer. 
When  the  staining  is  sufficient  the  nuclei  of  the  leucocytes,  when  examined 
under  the  microscope,  should  have  a  ruby-red  colour.  Then  decolorise 
slightly,  by  washing  in  alcohol  for  two  or  three  seconds,  or  in  water  for 
about  half  an  hour.  Then  allow  to  dry  and  mount  in  balsam. 

Second  Method.  —  Equal  parts  of  A  and  B  (say  500  c.c.  of  each)  are 
mixed,  allowed  to  stand  for  6-12  hours,  the  mixture  being  thoroughly 
stirred  from  time  to  time.  The  mixture  is  then  filtered,  and  the  deposit 
which  is  got  on  the  filter  is  dried  and  powdered.  A  .15  per  cent  solu- 
tion of  this  is  made  in  methyl  alcohol  (Merck,  "for  analysis").  This 
alcoholic  solution  fixes  and  stains  at  the  same  time.  Place  3-4  drops  on 
the  film  for  half  a  minute,  then  add  6-8  drops  of  distilled  water,  mix 
with  the  stain,  and  allow  to  remain  for  five  -minutes  longer.  (This  in- 
tensifies the  staining,  especially  the  red  tint.)  Then  wash  in  water,  allow 
to  dry,  and  mount  in  balsam,  or  simply  examine  in  water. 

In  the  Romanowsky  method,  the  chromatin  of  the  parasites  ought  to 
be  brilliant  red,  the  protoplasm  blue. 

It  is  to  be  noted  that  with  practically  all  the  methods  of  staining, 
better  results  are  obtained  when  the  blood  films  have  been  recently  made 
than  when  they  have  been  kept  for  some  time. 


APPENDIX    D. 

AMCEBIC   DYSENTERY. 

IN  a  previous  chapter  it  has  been  pointed  out  that  the  term  "  dysen- 
tery "  has  been  applied  to  a  number  of  conditions  of  different  etiology 
and  the  relations  of  bacteria  as  causal  agents  have  been  discussed  (vide 
p.  349).  We  shall  here  consider  that  variety  of  tropical1  dysentery 
which  is  believed  to  be  due  to  an  amoeba,  and  hence  often  known  as 
amoebic  dysentery. 

Amongst  the  early  researches  on  the  relation  of  organisms  to  dysen- 
tery probably  the  most  important  are  those  of  Losch,  who  noted  the 
presence  and  described  the  characters  of  amoebae  in  the  stools  of  a  per- 
son suffering  from  the  disease,  and  considered  that  they  were  probably 
the  causal  agents.  Further  observations  on  a  more  extended  scalje  were 
made  by  Kartulis  with  confirmatory  results,  this  observer  finding  the 
same  organisms  also  in  liver  abscesses  associated  with  dysentery.  The 
subject  was,  however,  complicated  by  the  fact  that  the  same  or  closely 
similar  organisms  had  been  previously  found  in  the  intestine  in  normal 
conditions  and  in  other  diseases  than  dysentery  (by  Cunningham  and 
Lewis  and  others),  and  additional  research  confirmed  these  results. 
Two  questions  thus  arose.  In  the  first  place,  Is  there  an  amoeba  pe- 
culiar to  dysentery  (amoeba  dysenteriae)  and  distinguishable  from  the 
amoebae  present  in  other  conditions?  In  the  second  place,  Is  this  organ- 
ism the  cause  of  the  disease?  Both  of  these  questions  may  now  be 
said  to  be  practically  answered  in  the  affirmative.  Further,  Councilman 
and  Lafleur,  working  in  Baltimore,  have  found  that  this  variety  of  dys- 
entery can  be  distinguished  from  other  forms,  not  only  by  the  presence 
of  amoeba,  but  also  by  its  pathological  anatomy.  The  intestinal  lesions, 
to  which  reference  is  made  below,  are  of  a  grave  character,  mortality  is 
relatively  high,  and  recovery,  when  it  occurs,  is  protracted  on  account 
of  the  extensive  tissue  changes.  The  results  of  these  observers  have 
been  confirmed  by  those  obtained  in  Egypt  by  Kruse  and  Pasquale,  who 
have  also  supplied  important  facts  regarding  the  pathogenic  effects  of 

1  The  term  tropical  is  misleading,  as  amoebic  dysentery  is  known  to  develop 
independently  in  temperate  regions,  cases  being  not  infrequent  in  Baltimore,  Phila- 
delphia, and  New  York,  in  the  United  States,  and  cases  have  also  been  reported  in 
Germany. 

2  M  529 


530 


APPENDIX   D. 


the  amoebae  when  inoculated  into  animals.     The  following  description  is 
chiefly  taken  from  the  monographs  of  the  four  writers  last  mentioned. 

Characters  of  the  Amoeba.  —  The  amoebae,  as  seen  in '  the  stools  of 
a  case  of  amoebic  dysentery,  are  rounded  or  somewhat  irregular  proto- 
plasmic masses,  usually  measuring  about  25  to  35  /x,  in  diameter,  though 
both  larger  and  smaller  forms  are  met  with. 

When  the  parasite  is  at  rest  it  has  a  more  or  less  rounded  shape ; 
the  protoplasm  is  finely  granular  and  of  refractile  appearance,  and  is 
without  differentiation  into  layers.  The  organism  may  show  sluggish 
amoebic  movements  at  the  ordinary  temperature,  but  these  become  much 
more  active  when  a  warm  stage  is  used.  When  they  occur,  the  amoeba 
shows  differentiation  into  a  central  granular  endoplasm  and  an  outer 
hyaline  layer  or  ectoplasm  which  is  very  thin  and  well  marked  off  from 
the  former.  The  blunt  processes  which  are  protruded  in  amoebic 

movement  are 
composed  of  the 
ectoplasm  (Fig. 
169,  a,b).  By  the 
amoebic  move- 
ments slow  loco- 
motion may  be 
produced.  The 
amoebae  often  show 
vacuoles  in  their 
substance,  and  may 
contain  numerous 


FIG.  169. —  Amoebae  of  dysentery. 


a  and  b,  amoebae  as  seen  in  the  fresh  stools,  showing  blunt  amce 
boid  processes  of  ectoplasm.     The  endoplasm  of  a  shows  a  nucleus,  three   red 
red  corpuscles,  and  numerous  vacuoles;  that  of  b,  numerous  red  corpuscles    fwViip}-) 

> 


tO 


annpar 
and  a  few  vacuoles.  > 

c,  an  amoeba  as  seen  in  a  fixed  film  preparation,  showing  a  small   undergo       digCS- 
rounded  nucleus  (Kruse  and  Pasquale).     x  600.  .•       \  i  -u 

tion),     also      bac- 

teria, etc.  There  is  a  single  nucleus  which  lies  in  the  central  part  of  the 
organism  and  usually  measures  about  6  to  8  //,  in  diameter.  It  is  round 
or  oval  and  contains  a  nucleolus.  In  the  living  condition  the  nucleus  is 
invisible  or  is  faintly  seen,  but  becomes  very  evident  on  the  addition  of 
acetic  acid,  etc.  The  amoebae  break  down  pretty  rapidly  outside  the 
body,  and  examination  of  the  dysenteric  stools  twenty-four  hours  after 
being  passed  usually  fails  to  detect  any  of  them.  It  is  only  on  one  or 
two  rare  occasions  that  the  process  of  division  of  the  amoebae  has  been 
observed  and  described. 

By  some  there  have  also  been  described  encysted  forms.  These  are 
of  smaller  size,  about  10  to  15  /x,  with  a  well-marked  capsule,  sometimes 
showing  a  double  contour  and  a  central  protoplasm  in  which  a  nucleus 
may  or  may  not  be  visible.  It  is  still  doubtful,  however,  whether  these 


AMOEBIC   DYSENTERY. 


531 


structures  really  constitute  a  stage  in  the  development  of  the  organism, 
as  direct  transformation  from  the  one  form  into  the  other  has  not  been 
observed. 

Distribution  of  the  Amoebae.  —  As  already  stated,  they  are  usually 
found  in  large  numbers  in  the  contents  of  the  large  intestine  in  tropical 
amoebic  dysentery.  They  also,  however,  penetrate  into  the  tissues, 
where  they  appear  to  exert  a  well-marked  action.  In  this  disease  the 
lesions  are  chiefly  in  the  large  intestine,  especially  in  the  rectum  and  at 
the  flexures,  though  they  may  also  be  present  in  the  lower  part  of  the 
ileum.  At  first  there  are  seen  local  swellings  on  the  mucous  surface, 
chiefly  due  to  a  sort  of  inflammatory  gelatinous  oedema  with  little 
leucocytic  infiltration ;  soon,  however,  the  mucous  membrane  becomes 
partially  ulcerated,  more  or  less  extensive  necrosis  of  the  subjacent 
tissues  occurs,  and  thus  gangrenous  sloughs  result.  The  ulcers  thus 
come  to  have  irregular  and  overhanging  margins,  and  the  excavation 
below  is  often  of  wider  extent  than  the  aperture  in  the  mucous  mem- 
brane. The  amoebae  are  found  in  the  mucous  membrane  when  ulcers 
are  being  formed,  but  their  most  characteristic  site  is  beyond  the  ulcer- 
ated area,  where  they  may  be  seen  penetrating  deeply  into  the  sub- 
mucous,  and  even  into  the  muscular  coats.  In  these  positions  they  may 
be  unattended  by  any  other  organisms,  and  the  tissues  around  them 
show  cedematous  swelling  and  more  or  less  necrotic  change  without 
much  accompanying  cellular  reaction,  beyond  a  certain  amount  of  swell- 
ing and  proliferation  of  the  connective-tissue  cells.  This  action  of  the 
amcebse  on  the  tissues  explains  the  character  of  the  ulcers  as  just  described. 
These  lesions  are  considered  by  Councilman  and  Lafleur  to  be  charac- 
teristic of  amoebic  dysentery. 

As  a  complication  of  this  form  of  dysentery  liver  abscesses  are  of 
comparatively  common  occurrence.  They  are  usually  single  and  of 
large  size,  sometimes  there  are  more  than  one,  and  occasionally  numerous 
small  ones  may  be  present.  The  contents  are  usually  a  thick  pinkish 
fluid  of  somewhat  slimy  consistence  and  are  largely  constituted  by 
necrosed  and  liquefied  tissue  with  admixture  of  blood  in  varying  amount. 
Microscopic  examination  shows  chiefly  necrosed  and  granular  cells  and 
debris -resulting  from  their  disintegration,  whereas  ordinary  pus  cor- 
puscles are  scanty  or  may  be  practically  absent.  In  such  abscesses 
associated  with  dysentery  the  amoebae  are  usually  to  be  found,  and  not 
infrequently  are  the  only  organisms  present,  no  cultures  of  bacteria  being 
obtainable  by  the  ordinary  methods  (Fig.  1 70).  They  are  most  numerous 
at  the  spreading  margin,  and  this  probably  explains  a  fact  pointed  out 
by  Manson,  that  examination  of  the  contents  first  removed  may  give  a 
negative  result,  while  they  may  be  detected  in  the  discharge  a  day  or  two 
later.  The  action  here  on  the  tissues  is  of  an  analogous  nature,  namely, 


532 


APPENDIX   D. 


a  necrosis  with  softening  and  partial  liquefaction,  attended  by  little  or 
no  suppurative  change.  The  amoebae  have  also  been  found  in  the  sputum 
when  a  liver  abscess  has  ruptured  into  the  lung,  as  not  very  infrequently 
happens. 

Relations  to  the  Disease.  —  It  may  be  stated  in  the  first  place  that 
satisfactory  cultures  of  these  amoebae  outside  the  body  have  not  been 
obtained.  Kartulis  announced  that  he  had  cultivated  the  organism  on 

straw  infusion,  but  it  is  now  recog- 
nised that  his  results  are  erroneous, 
the  amoebae  observed  by  him  being 
probably  derived  from  the  infusion 
itself.  In  fact,  everything  seems  to 
show  that  the  amoebae  in  their  usual 
form  rapidly  disintegrate  outside 
the  body,  and  it  is  still  unknown  in 
what  form  they  survive  and  lead 
to  the  propagation  of  the  disease. 
The  points  of  distinction  between 
the  amoeba  of  dysentery  and  the 
ordinary  amoeba  coli,  so  far  as  the 
FIG  170. -Section  of  wall  of  liver  ab-  morphology  is  concerned,  are  that 


the  latter  is  on  the  whole  of  smaller 


scess,  showing  an  amoeba  of  spherical  form 

with  vacuolated  protoplasm.     From  a  case 

published  by  Surgeon-major  D.G.  Marshall.     gize    itg   protoplasm   IS   more   finely 

X  1000. 

granular,  and  it  does  not  appear  to 

take  up  red  corpuscles,  etc.,  as  is  the  case  with  the  former.  The  dis- 
tinction, however,  can  only  be  definitely  drawn  by  means  of  experiment. 
Injections  of  certain  quantities  of  dysenteric  stools  containing  the  amoebae 
into  various  animals  per  rectum  have  been  carried  out  by  different  ob- 
servers, especially  by  Kruse  and  Pasquale.  In  cats,  in  the  majority  of 
cases,  a  haemorrhagic  enteritis  is  produced,  amoebae  being  present  in 
the  stools  and  also  invading  the  mucous  membrane  of  the  intestine  in  the 
ulcerated  areas  which  are  sometimes  formed.  The  deep  infiltration  of 
the  submucous  coat  by  the  amoebae,  which  is  so  characteristic  a  feature 
in  the  human  disease,  does  not  occur  in  these  animals.  Not  infrequently 
death  follows.  Kruse  and  Pasquale  obtained  corresponding  results  when 
the  material  from  a  liver  abscess,  containing  amoebae  without  any  other 
organisms,  was  injected.  In  the  absence  of  cultures  of  amoebae  outside 
the  body,  this  result  must  be  taken  as  strong  evidence  that  the  disease 
produced  in  cats  is  really  caused  by  the  amoebae.  Similar  injections 
with  material  containing  amoebae  derived  from  other  sources  are  unat- 
tended by  any  pathogenic  effects  of  similar  nature.  ,  Feeding  the  ani- 
mals with  material  containing  the  amoebae  is  much  more  uncertain  in  its 
effect.  Quincke  and  Roos  obtained  no  effects  when  the  amoebae  were 


AMCEBIC   DYSENTERY. 


533 


administered,  but  they  obtained  a  fatal  result  in  two  out  of  four  cases 
when  the  cyst-like  forms  were  given.  From  this  fact  they  infer  that  the 
latter  are  probably  a  cystic  stage  of  the  former,  and  that  the  former  are 
destroyed  in  the  gastric  contents.  This  practically  constitutes  the  only 
important  evidence  that  a  cystic  stage  of  the  organism  has  really  been 
observed.  These  observers  found  that  the  cyst-like  bodies  were  still 
present  even  after  the  material  had  been  kept  for  two  or  three  weeks. 

From  the  above  facts,  all  of  which  have  received  ample  confirma- 
tion with  the  exception  of  the  statements  regarding  the  cyst-like  forms, 
there  can  be  little  or  no  doubt  that  the  amoebae  described  are  the  causes 
of  the  form  of  dysentery  with  which  they  are  associated.  We  are  still  igno- 
rant, however,  as  to  their  life  history  outside  the  body,  and  the  modes  by 
which  infection  is  produced.  Further,  in  any  case  where  they  act  as 
the  primary  agent,  secondary  inflammatory  changes  in  the'  intestine  may 
be  produced  by  the  action  of  various  bacteria.  It  is  also  of  importance 
to  note  that  the  serum  of  patients  suffering  from  amoebic  dysentery  gives 
no  agglutinating  reaction  with  Shiga's  bacillus  of  dysentery  (vide  p.  349). 

Methods  of  Examination.  —  The  faeces  in  a  case  of  suspected  dys- 
entery ought  to  be  examined  microscopically  as  soon  as  possible  after 
being  passed,  as  the  amoebae  disappear  rapidly,  especially  when  the 
reaction  becomes  acid.  A  drop  is  placed  on  a  slide  without  the  addi- 
tion of  any  reagent,  a  cover-glass  is  placed  over  it  but  not  pressed  down, 
and  the  preparation  is  examined  in  the  ordinary  way  or  on  a  hot  stage, 
preferably  by  the  latter  method,  as  the  movements  of  the  amoebae 
become  more  active  and  it  is  difficult  to  recognise  them  when  they  are 
at  rest.  Hanging-drop  preparations  may  also  be  made  by  the  methods 
described.  Dried  films  are  not  suitable,  as  in  the  preparation  of  these 
the  amoebae  become  broken  down  ;  but  films  may  be  fixed  with  corrosive 
sublimate  or  other  fixative  (vide  p.  90).  In  sections  of  tissue  the  amoe- 
bae may  be  stained  by  methylene-blue,  by  safranin,  by  haematoxylin  and 
eosin,  etc.  Benda's  method  of  staining  with  safranin  and  light-green  is 
also  a  very  suitable  one.  Sections  are  stained  for  several  hours  in  a 
saturated  solution  of  safranin  in  aniline-oil  water  (p.  101),  they  are  then 
washed  in  water  and  decolorised  in  a  \  per  cent  solution  of  light  green 
in  alcohol  till  most  of  the  safranin  is  discharged,  the  nuclei,  however, 
remaining  deeply  stained.  In  this  method  the  nuclei  of  the  amoebae  are 
coloured  red  (like  those  of  the  tissue  cells),  the  protoplasm  being  of  a 
purplish  tint. 


BIBLIOGRAPHY. 

GENERAL  TEXT-BOOKS.  —  In  English  the  student  may  consult  the  follow- 
ing: "Micro-organisms  and  Disease,'1  E.  Klein,  3rd  ed.  London,  1896. 
"  Bacteriology  and  Infective  Diseases,"  Edgar  M.  Crookshank,  London,  1898. 
"A  Manual  of  Bacteriology,"  George  M.  Sternberg,  New  York,  ist  ed.  1893, 
2nd  ed.  1896  (this  bock  contains  a  full  bibliography).  "  Text-book  upon  the 
Pathogenic  Bacteria,"  Joseph  M'Farland,  Philadelphia,  1900.  "Practical  Bac- 
teriology," A.  A.  Kanthack  and  J.'H.  Drysdale,  London,  1895.  "  Bacteria  and 
their  Products,"  G.  S.  Woodhead,  London,  1891.  The  articles  on  bacterio- 
logical subjects  in  Clifford  Allbutt's  "  System  of  Medicine,"  London,  are  of 
the  highest  excellence,  and  have  full  bibliographies  appended.  For  the 
hygienic  aspects  of  bacteriology  see  "  System  of  Hygiene,"  Stevenson  and 
Murphy,  London,  1892-94.  Atlas  and  Principles  of  Bacteriology,  by  Lehmann 
and  Neumann  (trans,  by  G.  H.  Weaver),  Philadelphia,  1901. 

For  non-pathogenic  bacteria  occurring  in  connection  with  pathological 
work  consult  Heim,  op.  cit.  infra.  For  fungi  see  De  Bary,  "  Comparative 
Morphology  and  Biology  of  the  Fungi,  Mycetozoa  and  Bacteria,"1  trans,  by 
Garnsey  and  Balfour,  Oxford,  1887;  Sachs,  "Text-book  of  Botany,"  trans. 
by  Garnsey  and  Balfour,  Oxford,  1887,  ii. 

In  German  :  "  Die  Mikroorganismen,"  by  Dr.  C.  Fliigge,  3rd  ed.  Leipzig, 
1896.  (The  first  edition  of  this  book,  published  in  1886,  was  a  monograph 
by  Fliigge.  The  third  is  practically  a  new  work  edited  by  Fliigge  and 
written  by  Frosch,  Gotschlich,  Kolle,  Kruse,  and  R.  PfeifFer.  It  contains 
a  very  full  treatment  of  the  subject  and  has  a  complete  list  of  references.) 
"Lehrbuch  der  pathologischen  Mykologie,"  by  Baumgarten,  Braunschweig, 
1890.  "Grundriss  der  Bakterienkunde,"  C.  Fraenkel,  Berlin,  1890.  "Die 
Methoden  der  Bakterien-Forschung,"  F.  Hueppe,  Wiesbaden,  1891. 
"  Naturwissenschaftliche  Einfiihrung  in  die  Bakteriologie,"  F.  Hueppe, 
Wiesbaden.  1896.  "Einfuhrung  in  das  Studium  der  Bakteriologie,"  C. 
Gunther,  Leipzig,  1893  (4th  ed.  1895).  "Lehrbuch  der  bakteriologischen 
Untersuchung  und  Diagnostik,"  L.  Heim,  Stuttgart,  1894.  "System  der 
Bakterien  "  by  Migula,  Jena,  1900. 

In  French  :  Roger,  "  Les  maladies  infectieuses,"  Paris,  1902.  For  students, 
two  extremely  useful  books  are  "  Precis  de  microbie,"  by  Thoinot  and 
Masselin,  3rd  ed.  Paris,  1896,  and  "  Precis  de  bacteriologie  clinique,"  Wurtz, 
Paris,  1895. 

PERIODICALS.  — For  references  to  current  work  see  Centralbl.  f.  BakterioL 
M.  Parasitenk.,  Jena.  This  publication  commenced  in  1887.  Two  volumes 
are  issued  yearly.  In  1895  it  was  divided  into  two  parts.  Abtheilung  I. 
deals  with  Medizinisch-hygienische  Bakteriologie  und  thierische  Par asitenkunde. 

534 


BIBLIOGRAPHY. 


535 


The  volumes  of  this  part  are  numbered  consecutively  with  those  of  the  former 
series,  the  first  issued  thus  being  vol.  xvii.  Commencing  in  1902  with  volume 
xxxi.,  each  volume  of  Abtheilung  I.  was  further  divided  into  two  parts,  one 
consisting  of  Originate  the  other  of  Referate.  Abtheilung  II.  deals  with 
Allgemeine  landwirtschaftlich-technologische  Bakteriologie,  Garungs-physiologie 
und  Pflanzenpathologie.  The  first  volume  is  entitled  Zweite  Abtheilung,  Bd. 
I.  It  contains  original  articles.  Referate,  etc. 

The  most  complete  account  of  the  work  of  the  year  is  found  in  \hsjahresb. 
u.  d.  Fortschr.  .  .  .  d.  path.  Mikroorganismen,  conducted  by  Baumgarten. 
and  published  in  Braunschweig.  This  publication  commenced  in  1887.  Its 
disadvantage  is  that  the  volume  for  any  year  does  not  usually  appear*  till  two 
years  later. 

Bacteriology  is  also  dealt  with  in  the  Index  Medicus  and  Bibliographia 
Medica.  For  valuable  lists  of  papers  by  particular  authors  see  Royal  Society 
Catalogue  of  Scientific  Papers;  and  Index. Catalogue,  Library  of  Surg.-Gen. 
U.  S.  Army,  Series  II,  Washington. 

The  chief  bacteriological  periodicals  are  \\\z  Journ.  Path,  and  Bacterial., 
Edinburgh  and  London,  edited  by  Sims  Woodhead  ;  the  Ztschr.  f.  Hyg.  u. 
Infectionskrankh.,  Leipzig,  edited  by  Koch  and  Fliigge,  and  the  Ann.  de  FInst. 
Pasteur,  Paris,  edited  by  Duclaux  ;  Journ.  Exper.  Med.,  Baltimore,  edited  by 
Welch  ;  Journ.  Hyg.,  Cambridge,  edited  by  Nuttall ;  Journ.  Med.  Research, 
Boston,  edited  by  H.  C.  Ernst. 

Valuable  papers  also  from  time  to  time  appear  in  the  Lancet,  Brit.  Med. 
Journ.,  Bull.  Johns  Hopkins  Hosp.,  Reports  of  the  Johns  Hopkins  Hosp.,  Univ. 
Pennsyl.  Med.  Bull.,  Deutsche  med.  Wchnschr.,  Berl.  klin.  IVchnschr.,  Semaine 
med.,  Arch.  f.  Hyg.,  Arch.  f.  exper.  Path.  u.  Pharmakol.  Besides  these  peri- 
odicals the  student  may  have  to  consult  the  Reports  of  the  Med.  Off.  of  the 
Local  Government  Board,  which  contain  the  reports  of  the  medical  officers, 
also  the  Proc.  Roy.  Soc.  London,  the  Conipt.  rend.  Acad.  d.  sc.,  Paris,  the 
Compt.  rend.  Soc.  de  biol,  Paris,  and  the  Arb.  a.  d.  k.  Gsndhtsamte.  (the  first 
two  volumes  of  the  last  were  denominated  Mittheilungen} . 

CHAPTER   I.  —  GENERAL  MORPHOLOGY  AND  BIOLOGY. 

Consult  here  especially  Fliigge,  "Die  Mikroorganismen."  De  Bary, 
"Bacteria,1'  translated  by  Garnsey  and  Bayley  Balfour,  Oxford,  1887. 
Zopf,  "Zur  Morphologic  der  Spaltprlanzen,11  Leipzig,  1882;  "  Beitr.  z. 
Physiologic  und  Morphologic  nieclerer  Organismen,11  5th  ed.,  Leipzig,  1895. 
Cohn,  Beitr.  z.  Biol.  d.  Pflanz.,  Bresl.  (1876),  ii.  v.  Nageli,  "Die  niederen 
Pilze,"  Munich,  1877;  "  Untersuchungen  iiber  niedere  Pilze,"  Munich, 
1882.  Migula,  "System  der  Bakterien,"  Jena,  1900.  Duclaux,  "Traite' 
de  microbiologie,"  Paris,  1898-99.  For  general  morphological  relations  see 
Marshall  Ward,  art.  "  Schizomycetes,"  Encyc.  Brit.,  gth  ed.,  xxi.  398  :  xxvi. 
51.  Engler  u.  Prantl,  "  Die  natiirlichen  Pflanzenfamilien,"  Lieferung 
129.  — "  Schizophyta"  (by  W.  Migula).  STRUCTURE  OF  BACTERIAL  CELL. 
—  Butschli,  "  Uber  den  Bau  der  Bakterien,"  Leipzig,  1890;  "  Weitere 
Ausfiihrungen  iiber  den  Bau  der  Cyanophyceen  und  Bakterien,"  Leipzig, 
1896.  Fischer,  op.  cit.  in  text.  Buchner,  Longard  u.  Riedlin,  Centralbl. 


536  BIBLIOGRAPHY. 

f.  Bakteriol.  u.  Parasitenk.,  ii.  i.  Ernst,  Ztschr.  f.  Hyg.,\.  428;  Babes, 
ibid.,  v.  173.  Neisser,  ibid.,  iv.  165.  Nakanishi,  Centralbl. f.  Bakt.  u.  Para- 
sitenk., Abt.  I.,  Bd.  xxx.,  Nos.  4,  5,  and  6.  MOTILITY.  —  Klein,  BUtschli, 
Fischer,  Cohn,  loc.  cit.  Loffler,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  vi.  209  ; 
vii.  625.  PIGMENTS.  —  Zopf,  loc.  cit. ;  Galeotti,  Ref.  in  Centralbl.  f.  Bakteriol. 
u.  Parasitenk.,  xiv.  696.  Babes,  Ztschr.  f.  Hyg.,xx.  3.  SPORULATION. — 
Prazmowski,  BioL  Centralbl.,  viii.  301.  A.  Koch,  Botan.  Ztg.,  (1888)  Nos. 
18-22.  Buchner,  Sitzungsb.  d.  math.-phys.  Cl.  d.  k.  bayer.  Akad.  d.  Wissensch. 
zu  Milnchen,  7th  Feb.  1880.  R.  Koch,  Mitth.  a.  d.  k.  Gsndhlsamte.,  i.  65. 
CHEMICAL  STRUCTURE  OF  BACTERIA.  —  Nencki,  Ber.  d.  deutsch.  Chem. 
Gesellsch.,  (1884)  xvii.  2605.  Cramer,  Arch.  f.  Hyg.,  xvi.  154.  Buchner, 
Berl.  klin.  Wchnschr.,  (1890)  673,  1084;  vide  Fliigge,  op.  cit.  CLASSIFICA- 
TION OF  BACTERIA.  —  For  general  review  see  Marshall  Ward,  Ann.  of  Botany, 
vi.  103;  Migula,  loc.  cit.  supra.  Lachner-Sandoval,  Ueber  Strahlenpilze 
[Inaug.  Dissert^\,  Strassburg,  1898.  FOOD  OF  BACTERIA.  —  Nageli,  Cohn,  op. 
cit.  Pasteur,  "Etudes  sur  la  biere,"  1876.  Hueppe,  Mitth.  a.  d.  k.  Gsndhts- 
amte.,  ii.  309.  RELATIONS  TO  OXYGEN.  —  Pasteur,  Compt.  rend.  Acad.  d.  sc., 
Hi.  344,  1142  ;  Kitasato  u.  Weyl,  Ztschr.  f.  Hyg.,  viii.  41,  404  ;  ix.  97.  TEM- 
PERATURE. —  Vide  Fliigge,  op.  cit.  ;  for  thermophilic  bacteria,  Rabinowitsch, 
Ztschr.  f.  Hyg.,x\.  154;  Macfadyen  and  Blaxall,y<9//r;/.  Path,  and  Bacteriol., 
iii.  87.  ACTION  OF  BACTERIAL  FERMENTS.  —  Salkowski,  Zlschr.f.  BioL,  N.F. 
vii.  92  ;  Pasteur  et  Joubert,  Compt.  rend.  Acad.  d.  sc.,  Ixxxiii.  5  ;  Sheridan  Lea,. 
Journ.  Physiol.,  vi.  136;  Beijerinck,  Centralbl.  f.  Bacteriol.  u.  Parasitenk., 
Abth.  II.  i.  221  ;  E.  Fischer,  Ber.  d.  deutsch.  chem.  Gesellsch.,  xxviii.  1430; 
Liborius,  Ztschr.  f.  Hyg.,  i.  115  ;  see  also  Pasteur,  "Royal  Society  Catalogue 
of  Scientific  Papers.1'  VARIABILITY.  —  Cohn,  Nageli,  Fliigge,  op.  cit. 
Winogradski,  "  Beitr.  z.  Morph.  u.  Physiol.  d.  Bakt.,"  Leipzig,  1888;  Ray 
Lankester,  Quart.  Journ.  Micr.  Sc.,  N.S.  (1873)  xiii.  408;  (1876)  xvi.  27,  278. 
NITRIFYING  ORGANISMS.  —  Winogradski,  Ann.  de  I"1  hist.  Pasteur,  iv.  213,257, 
760;  v.  92,  577.  Maze,  ibid.,  xi.  44;  xii.  i,  263. 

CHAPTER   II.  — METHODS  OF  CULTIVATION  OF  BACTERIA. 

FOR  GENERAL  PRINCIPLES.  — Pasteur,  Compt.  rend.  Acad.  d.  sc.,  1.  303; 
Ii.  348,  675  ;  Ann.  de  chem.,  Ixiii.  5  ;  Tyndall,  "  Floating  Matter  of  the  Air  in 
Relation  to  Putrefaction  and  Infection,"  London,  1881;  H.  C.  Bastian,  "The 
Beginnings  of  Life,1'  London,  1872.  METHODS  OF  STERILISATION.  —  R. 
Koch,  Gaffky,  and  Loffler,  Mitth.  a.  d.  k.  Gsndhtsamte.,  i.  322;  Koch  u. 
Wolffhiigel,  ibid.,  i.  301.  CULTURE  MEDIA.  —  See  text-books,  especially 
Kanthack  and  Drysdale  ;  Pasteur,  "  Etudes  sur  la  biere,"  Paris,  1876 ;  R.  Koch, 
Mitth.  a.  d.  k.  Gsndhtsamte.,  i.  i  ;  Roux  et  Nocard,  Ann.  de  rinst.  Pasteur, 
i.  i  ;  Roux,  ibid.,  ii.  28;  Durham,  Jour.  Exper.  Med.,  1901,  v.  353;  Marmo- 
rek,  Ann.  de  Vlnst.  Pasteur,  ix.  593  ;  Hiss,  Science,  (1902)  Mar.  7.  Kitasato 
u.  Weyl,  op.  cit.  supra  ;  P.  and  Mrs.  Percy  Frankland,  "  Micro-organisms  in 
water,"  London,  1894.  Fuller,  Rep.  Amer.  Pub.  Health  Ass.,  xx.  381.  Theo- 
bald Smith,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,\\\.  502;  xiv.  864.  Dur- 
ham, Brit.  Med.  Journ.,  1898,  i.  1387.  "Report  of  American  Committee 
on  Bacteriological  Methods,"  Concord,  1898.  Park,  Centralbl.  f.  Bakt.  u. 


BIBLIOGRAPHY.  537 

Parasitenk.,  Abt.  I.  Bd.  xxix.  445.  Wright,  Journ.  Boston  Soc.  Med.  Sc., 
(1900)  iv.  119.  Hill,  Journ.  Med.  Research,  (1902)  n.  s.  II.  202.  Harris, 
Johns  Hopkins  Hasp.  Bull.,  (1902)  No.  134,  112.  Hill,  Journ.  Boston  Soc. 
Med.  Sc.,  (1899)  Jan.  Th.  Smith,  Wilder  Quart.  Cent.  Book,  (1893)  187. 
Novy,  Laboratory  Work  in  Bacteriology,  2nd  ed.,  1899. 

CHAPTER  III.  —  MICROSCOPIC  METHODS,  ETC. 

Consult  text-books,  especially  Klein,  Kanthack  and  Drysdale,  Hueppe, 
Giinther,  Heim,  Thoinot  et  Masselin ;  also  Bolles  Lee,  u  The  Microtomisf  s 
Vademecum,"  5th  ed.,  London,  1900  (this  is  the  most  complete  treatise  on 
the  subject).  Rawitz,  op.  cit.  in  text;  Koch,  Mitth.  a.  d.  k.  Gsndhtsamte., 
i.  i  ;  Ehrlich,  Ztschr.f.  klin.  Med.,  i.  553  ;  ii.  710.  Gram,  Fortschr.  d.  Med. 
(1884)  ii.  No.  6;  Nicholle,  Ann.  de  rinst.  Pasteur,  ix.  666;  Kiihne,  "Prak- 
tische  Anleitung  zum  mikroscopischen  Nachweis  der  Bakterien  im  tierischen 
Gewebe,"  Leipzig,  1888  ;  Van  Ermengem,  ref.  Centralbl.f.  Bakteriol.  u.  Para- 
sitenk., xv.  969;  Kendall,  Journ.  App.  Microscopy,  v.  No.  6,  1836.  Richard 
Muir,  Journ.  Path,  and  BacterioL,  v.  374  ;  Mann,  "  Physiological  Histology,'' 
Oxford,  1902. 

AGGLUTINATION.  —  Dele'pine,  Brit.  Med.  Journ.,  (1897)  ii.  529,  967. 
Widal  et  Sicard,  Ann.  de  FInst.  Pasteur,  xi.  353.  Wright,  Brit.  Med. 
Journ.,  (1897)  i.  139;  (1898)  i.  355. 

CHAPTER   IV.  — BACTERIA  OF  AIR,  SOIL,  WATER.     ANTISEPTICS. 

AIR,  SOIL,  AND  WATER.  —  Petri,  Ztsch.f.  Hyg.,  iii.  i  ;  vi.  233.  Sedgwick 
and  Tucker,  Proc.  Soc.,  Arts  Mass.  fnst.  Tech.,  (1887-8)  51,  (Boston).  Tucker, 
Twentieth  Annl.  Rept.  State  Bd.  Health  of  Mass.,  (1888)  1 6 1 .  Fliigge,  Ztsch . 
f.  Hyg.,  xxv.,  179.  Sticher,  ibid.,  xxx.,  163.  Weyl,  "  Handbuch  der  Hygiene," 
Jena,  1896,  et  seq.  Houston,  Rep.  Med.  Off.  Local  Gov.  Bd.,  xxvii.  (1897-98) 
251  ;  xxviii.  (1898-99)  413, 439, 467  ;  xxix.  (1899-1900)  458,  489.  Stone,  Amer. 
Med.,  (1902)  Jan.  25,  154.  MacConkey,  Thomp.  Yates  Lab.  Repts.,  (1900) 
III.  i.  41  ;  ibid.,  (1901)  III.  ii.  151.  MacConkey  and  Hill,  ibid.,  (1901)  IV. 
i.  151.  Grlinbaum  and  Hume,  Brit.  Med.  Journ.,  (1902)  June  14,  1473. 
Sidney  Martin,  ibid.,  xxvi.  (1896-97)  231;  xxvii.  (1897-98)  308;  xxviii. 
(1898-99)  382.  Horrocks,  "  Bacteriological  Examination  of  Water,"  London, 
1901.  Percy  and  G.  C.  Frankland,  "  Micro-organisms  in  Water,1' London, 
1894.  Dibdin,  "Purification  of  Sewage  and  Water,"  London,  1897.  Ann. 
Rep.  Bd.  Health,  Mass.,  Boston,  1890,  et  seq. 

ANTISEPTICS.  —  R.  Koch,  Mitth.  a.  d.  k.  Gsndhtsamte.,  i.  234.  Behring, 
Ztsch.f.  Hyg.,  ix.  395.  Ritchie,  Trans.  Path.  Soc.,  London,!.  256.  Rideal, 
"  Disinfection  and  Disinfectants,"  London,  1898. 

CHAPTER  V.  —  FUNGI:  NON-PATHOGENIC  AND  PATHOGENIC. 

For  NON-PATHOGENIC  bacteria  usually  occurring  in  man  consult  Heim,  op. 
cit.  For  fungi  see  De  Bary,  "  Comparative  Morphology  and  Biology  of  the 
Fungi,  Mycetozoa  and  Bacteria,"  trans,  by  Garnsey  and  Balfour,  Oxford,  1887; 


538  BIBLIOGRAPHY. 

Sachs,  "Text-book  of  Botany,"  trans,  by  Garnsey  and  Balfour,  Oxford,  1887, 
ii.  PATHOGENIC  FUNGI.  —  Rolleston  in  Allbutfs  Syst.  of  Med.,  1899.  Pear- 
son and  Ravenel,  Univ.  Med.  Mag.,  (Philadelphia)  1900,  Aug.  Busse,  Cen- 
tralbl. f.  Bakt.  u.  Parasitenk.,  (1894)  xvi.  175.  Virchow,  Archiv,  (1895) 
cxl.  23  ;  ibid.,  (1896)  cxliv.  360.  "Die  Hefen  als  Kranksheitserreger," Berlin, 
1897  (Hirschwald).  Buschke,  Volkmann's  Samml.  Klin.  Vortdge,  (1898) 
No.  218.  Verhand.  d.  Deutsch.  Dermat.  Gesellsch.,  VI.  Congr.,  (1899)  181. 
Gilchrist,  Johns  Hopkins  Hosp.  Repts.,  (1896)  i.  269.  Gilchrist  and  Stokes, 
Journ.  Exper.  Med.,  (1898)  iii.  53.  Schenck,  Johns  Hopkins  Hosp.  Bull., 
(1898)  ix.  286.  Ophiils  and  Moffitt,  Philadel.  Med.  Journ.,  (1900)  June 
30.  Montgomery,  Brit.  Journ.  Dermat.,  xii.  No.  144.  Ricketts,  Journ. 
Med.  Research,  (1901)  vi.  No.  3.  Hektoen,/07/r#.  Exper.  Med.,  (1899)  iv. 
261.  Hektoen  and  Perkins,  ibid.,  (i^oo)  V.  i.  77. 


CHAPTER  VI.  —  RELAT IONS  OF  BACTERIA  TO  DISEASE,  ETC. 

As  the  observations  on  which  this  chapter  is  based  are  scattered  through 
the  rest  of  the  book,  the  references  to  them  will  be  found  under  the  different 
diseases. 


CHAPTER  VII.  —  INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS. 

Ogston,  Brit.  Med.  Journ.,  (1881)  i.  369.  Rosenbach,  "  Mikroorgan- 
ismen  bei  den  Wundinfectionskrankheiten  des  Menschen,"  Wiesbaden,  1884. 
Passet,  Fortschr.  d.  Med.,  (1885)  Nos.  2  and  3.  Welch,  Trans.  Congr.  Ainer. 
Phys.  and  Surg.,  (1891)  ii.  ;  Maryland  Med.  Journ.,  (1891)  Nov.  14.  W. 
Watson  Cheyne,  "Suppuration  and  Septic  Diseases,"  Edinburgh,  1889. 
Grawitz,  Vir chow's  Archiv,  cxvi.  116;  Deutsche  med.  Wchnschr.,  (1889)  No. 
23.  Steinhaus,  Ztschr.  f.  Hyg.,  v.  518  (micrococcus  tetragenus)  ;  "Die 
Aetiologie  der  acuten  Eiterung,"  Leipzig,  1889.  Christmas-Dirckinck-Holm- 
feld,  "Recherches  experimentales  sur  la  suppuration,"  Paris,  1888.  Muir, 
Journ.  Path,  and  Bacteriol,  vii.  161  ;  Trans.  Path.  Soc.  London,  1902.  Carre, 
Fortschr.  d.  Med.,  (1885)  No.  6.  Marmorek,  Ann.de  Flnst.  Pasteur,  ix.  593. 
Petruschky,  Ztschr.  f.  Hyg.,  xvii.  59;  xviii.  413;  xxiii.  142;  (with  Koch, 
xxiii.  477).  Liibbert,  "  Biologische  Spaltpilzuntersuchung,"  Wurzburg,  1886. 
Krause,  Fortschr.  d.  Med.,  (1884)  Nos.  7  and  8.  Ribbert,  Fortschr.  d.  Med., 
(1886)  No.  i.  Widal  et  Besancon,  Ann.  de  Plnst.  Pasteur,  ix.  104.  V.  Lin- 
gelsheim",  Ztschr.  f.  Hyg.,  x.  331  ;  xii.  308.  Behring,  Centralbl.  f.  Bakteriol. 
u.  Parasitenk.,  xii.  192.  Thoinet  et  Masselin,  Rev.  de  med.,  (1894)  449. 
Orth  u.  Wyssokowitsch,  Centralbl.  f.  d.  med.  IVissensch.  (1885)  577.  Netter, 
Arch,  de  physiol.  norm,  et  path.,  (1886)  106.  Weichselbaum,  Wien.  med. 
Wchnschr.,  (1885)  No.  41;  (1888)  Nos.  28-32;  Centralbl.  f.  Bakteriol.  u. 
Parasitenk.,  ii.  209;  Beitr.  2.  path.  Anat.  u.  2.  allg.  Path.,  iv.  127.  Becker, 
Deutsche  med.  Wchnschr.,  (1883)  No.  46.  Lannelongue  et  Achard,  Ann.  de 
rinst.  Pasteur,  v.  209.  Fehleisen,  "  Die  Aetiologie  des  Erysipels,"  Berlin, 
1883.  Welch,  Am.  Journ.  Med.  Sc.,  1891,  439.  Lemoine,  Ann.  de  r hist. 
Pasteur,  ix.  877.  Kurth,  Arb.  a.  d.  k.  Gsndhtsamte.,  vii.  389.,  Knorr,  Ztschr. 
f.  Hyg.,  xiii.  427.  Bulloch,  Lancet,  (1896)  i.  982,  1216.  Bordet,  Ann.  de 


BIBLIOGRAPHY.  539 

r/nst.  Pasteur,  xi.  177.  Booker  (streptococcus  enteritis),  Johns  Hopkins 
Hosp.  Rep.,  vi.  159.  Hirsch,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  xxii. 
369.  Libman,  ibid.,  xxii.  376.  Gilchrist,  Contrib.  to  the  Sc.  of  Med.,  dedi- 
cated by  his  pupils  to  William  H.  Welch,  Baltimore,  (1900)  409.  MacCal- 
lum  and  Hastings,  Journ.  Exper.  Med.,  (1899)  iv.  521 .  Harris  and  Longcope, 
Centralbl.  f.  Bakt.  u.  Parasitenk.,  Abt.  i.  Bd.  xxx.  No.  9. 

Meningitis.  —  Weichselbaum,  Fortschr.  d.  Med.,  (1887)  v.  573,  620. 
Jaeger,  Ztschr.f.  Hyg.,  xix.  351.  Councilman,  Mallory,  and  Wright,  "Epi- 
demic Cerebro-spinal  Meningitis,11  Rep.  Bd.  Health  Mass.,  Boston,  1898  (full 
references).  Gwyn,  Johns  Hopkins  Hosp.  Bull.,  (1899)  109. 

Conjunctivitis.  —  Morax,  Ann.  de  VInst.  Pasteur,  (1896)  x.  337.  Eyre, 
Journ.  Path,  and  BacterioL,  vi.  I.  Miiller,  Wien.  med.  Wchnschr.,  1897. 
Axenfeld,  "  Ergebnisse  der  Allgem.  Pathol.  u.  Path.  Anat."  (Lubarsch  u. 
Ostertag)  1901  (full  references). 

Acute  Rheumatism.  —  Triboulet  et  Coyon, .  Bull.  Soc.  med.  d.  hop.  de 
Paris,  (1898)  93.  Westphal,  Wassermann  u.  Malkoff,  Berl.  klin.  Wchnschr., 
(1899)  638.  Poynton  and  Paine,  Lancet,  (1900)  ii.  861,  932  (full  references). 
Achalme,  Archiv.  de  Med.  Exper.,  (1898)  xi.  370;  Gaz.  Med.  de  Paris, 
(1901)  Apr.  6.  Thiroloix,  Sent,  med.,  (1896)  376,^420,  945.  Bettencourt, 
Archiv.  de  Med.,  ii.  298.  Hewlett  (read  before  Chelsea  Clin.  Soc.),  1901. 
G-wy\i,Johns  Hopkins  Hosp.  Bull.,  (1900)  xi.  185.  Foulerton  and  Rist,  Lan- 
cet, (1901)  553.  Poynton  and  Paine,  Lancet,  (1901)  May  4,  July  13,  Aug.  24; 
Centralbl.  f.  Bakt.  u.  Parasitenk.,  Abt.  i.  Bd.  xxxi.  502.  Singer,  Wien 
(Braunmiiller),  1898 ;  Wiener  Klin.  Woch.,  (1901)  No.  20,  482.  Meyer, 
Deutsch.  med.  Woch.,  (1901)  Feb.  7.  Menzer,  ibid.,  (1901)  Feb.  14.  Allaria, 
Riv.  crit.  di  elm.  Med.,  (1901)  Nov.  23;  (ref.  Brit.  Med.  Journ.,  1902, 
Jan.  ii). 

CHAPTER  VIII.  —  INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS, 
CONTINUED:  ACUTE  PNEUMONIAS. 

Sternberg,  Natnl.  Board  of  Health  Rept.,  Washington,  (1881)  74,  75. 
Pasteur,  £////.  de  rAcad.  de  Med.,  (1881)  Jan.  18  and  25,  Feb.  i  and  8.  Mar.  22 
and  29.  Friedlander,  Fortschr.  d.  Med.,\.  No.  22  ;  ii.  287  ;  Virchows  Archiv, 
Ixxxvii.  319.  A.  Fraenkel,  Ztschr.  f.  klin.  Med.  (1886)  401.  Salvioli  u. 
Zaslein,  Centralbl.  f.  d.  med.  Wissench.,  (1883)  721.  Ziehl,  ibid.,  (1883) 
433  ;  (1884)  97.  Klein,  ibid.,  (1884)  529.  Jiirgensen,  Berl.  klin.  Wchnschr., 
(1884)  270.  Seibert,  ibid.,  (1884)  272,  292.  Senger,  Arch.  f.  exper.  Path, 
u.  Pharmakol,  (1886)  389.  Weichselbaum,  Wien.  med.  Wchnschr.,  xxxvi. 
1301,  1339,  1367;  Monatschr.  f.  Ohrenh.,  (1888)  Nos.  8  and  9;  Centralbl. 
f.  Bakteriol.  u.  Parasitenk.,  v.  33.  Prochaska,  Centralbl.  f.  inner.  Med., 
Bd.  xxi.  No.  46  ;  Deutsch.  Arch.f.  kl.  Med.,  Bd.  Ixx.  Cole,  Johns  Hopkins 
Hosp.  Bull.,  (1902)  Nos.  135,  136.  Gamaleia,  Ann.  de  VInst.  Pasteur,  ii.  440. 
Guarnieri,  Atti  d.  r.  Accad.  med.  di  Roma,  (1888)  ser.  ii.  iv.  Kruse  u. 
Pansini,  Ztschr.  f.  Hyg.,  xi.  279.  E.  Fraenkel  u.  Reiche,  Ztschr.  f.  klin. 
Med.,  xxv.  230.  Sanarelli,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  x.  817. 
Lannelongue,  Gaz.  d.  hop.,  (1891)  379.  Netter,  Bull,  et  mem.  Soc.  med.  d. 
hop.  dc  Paris,  (1889);  Compt.  rend.  Acad.  d.  sc.,  (1890);  Compt.  rend. 


540  BIBLIOGRAPHY. 

Sac.  de  biol.,  Ixxxvii.  34.  G.  u.  F.  Klemperer,  Berl  klin.  Wchnschr., 
(1891)  893,  869.  Foa  u.  Bordoni-Uffreduzzi,  Deutsche  med.  Wchnschr., 
(1886)  No.  33.  Emmerich,  Munchen.  Died.  Wchnschr.,  (1891)  No.  32. 
Issaeff,  Ann.  de  Plnst.  Pasteur,  vii.  260.  Grimbert,  Ann.  de  Vlnst.  Pasteur, 
xi.  840.  Washbourn,  Brit.  Med.  Journ.,  (1897)  i.  510;  (1897)  ii.  1849. 
Eyre  and  Washbourn,  Journ.  Path,  and  Bacterial.,  iv.  394;  v.  13.  See  also 
Brit.  Med.  Journ.,  (1901)  ii.  760. 

CHAPTER  IX.  —  GONORRHOEA,  SOFT  SORE,  SYPHILIS. 

GONORRHCEA.  —  Neisser,  Centralbl.  f.  d.  med.  Wissensch.,  (1879)  497; 
Deutsche  med.  Wchrischr.,  (1882)  279;  (1894)  335.  Bumm,  u  Der  Mikro- 
organismus  der  gonorrhoischen  Schleimhauterkrankungen,"  Wiesbaden,  1885, 
2nd  ed.  1887  ;  Munchen.  med.  Wchnschr.,  (1886)  No.  27  ;  (1891)  Nos.  50  and 
51  ;  Centralbl.  f.  Gyndk.,  (1891)  No.  22;  Wien.  med.  Presse,  (1891)  No.  24. 
Bockhart,  Monatsh.  f.  prakt.  Dermat.,  (1886)  v.  No.  4;  (1887)  vi.  19. 
Steinschneider,  Berl.  klin.  Wchnschr.,  (1890)  No.  24;  (1893)  No.  29;  Ver- 
handl.  d.  deutsch.  der  mat.  Gesellsch.I.  Congress,  Wien,  1889,  159.  Wertheim, 
Wien.  klin.  Wchnschr.,  (1890)  25  ;  Deutsche  med.  Wchnschr.,  (1891)  No.  50  ; 
Arch.f.  Gynaek.,  xli.  Heft  i;  Centralbl.  f.  Gyndk.,  (1891)  No.  24;  (1892) 
No.  20  ;  Wien.  klin.  Wchnschr.,  (1894)  441.  Gerhardt,  Charite-Ann.,  (1889) 
241.  Leyden.  Ztschr.  f.  klin.  Med.  xxi.  607;  Deutsche  med.  Wchnschr., 
(1893)  909.  Bordoni-Uffreduzzi,  ibid.,  (1894)  484.  Councilman,  Am.  Journ. 
Med.  Sc.,  cvi.  277.  Finger,  Ghon,  u.  Schlagenhaufer,  Arch.  f.  Dermat.  u. 
Syph.,  xxviii.  3,  276.  Lang,  ibid.,  (1892)  1007;  Wien.  med.  Wchnschr., 
(1891)  No.  7;  "Der  Venerische  Katarrh,  dessen  Patholgie  und  Therapie," 
Wiesbaden,  1893.  Klein,  Monatschr.  f.  Geburtsh.  u.  Gynaek.,  (1895)  33. 
Michaelis,  Ztschr.  f.  klin.  Med.  xxix.  556.  Heiman,  New  York  Med.  Rec., 
(1895)  769,  (1896)  Dec.  19.  Foulerton,  Trans.  Brit.  hist.  Preven.  Med.,  i. 
40.  Frank,  Med.  News,  (1895)  Oct.  19.  Meija,  These  de  Paris,  (ref.  Cen- 
tralbl. f.  all.  Path.  u.  path.  Anat.,  1897,  Bd.  xii.  83).  Cushing,/0te  Hop- 
kins Hosp.  Bull.,  (1899)  No.  98,  75.  Hunner  and  Harris,  ibid.,'(  1902)  No. 
135,  121.  De  Christmas,  Ann.  deTInst.  Pasteur,  xi.  609.  Nicolaysen,  Cen- 
tralbl. f.  Bakteriol.  u.  Parasitenk.,  xxii.  305.  Rendu,  Berl.  klin.  Wchnschr. 
(1898)  431.  Wassermann,  Ztschr.  f.  Hyg.,  xxvii.  298;  Munchen.  med. 
Wchnschr.,  1901,  No.  8.  Lenhartz,  Berl.  klin.  Wchnschr.,  1897,  1138, 
Thayer  and  Lazear,  Journ.  Exper.  Med.,  iv.  81.  Wilson,  New  York  Univ. 
Bull.  Med.  Sc.,  (1901)  i.  No.  3.  Harris  and  Johnston,  Johns  Hopkins  Hosp. 
Bull.,  (1902)  No.  139,  236.  Konig,  Berl.  klin.  Wchnschr.,  1901,  No.  47. 
De  Christmas,  Ann.  de  rinst.  Pasteur,  (1900)  xiv.  331.  Raskai,  Deutsche 
med.  Wchnschr.,  1901,  No.  r.  Jundell,  Centralbl.  f.  Bakteriol.  u.  Parasi- 
tenk., xxix.  224.  Colombini,  ibid.^  xxiv.  955. 

SOFT  SORE.  —  Ducrey,  Monatsh.  f.  prakt.  Dermat.,  ix.  221.  Krefting, 
Arch.f.  Dermat.  u.  Syph.,  (1892)  263.  Jullien,  Journ.  d.  mal.  cutan.  et 
syph.,  (1892)  330.  Unna,  Monatsh.  /.  prakt.  Dermat.,  (1892)  475  ;  (1895) 
61.  Quinquand,  Semaine  med.,  (1892)  278.  Petersen,  Centralbl.  f.  Bakteriol. 
u.  Parasitenk.,  xiii.  743;  Arch.f.  Dermat.  u.  Syph.,  (1894)  419.  Audrey, 
Monatsh.  f.  prakt.  Dermat.,  (1895)  267.  Colombini,  Centralbl.  f.  Bakteriol. 


BIBLIOGRAPHY.  541 

u.  Parasitenk.,  xxv.  254.     Besan9on,  Griffon,  et  le  Sourd,  Annal.  de  dermat. 
et  syph.,  1901. 

SYPHILIS.  —  Lustgarten,  Wien.  med.  Wchnschr.,  (1884)  No.  47.  Doutre- 
lepont  u.  Schiitz,  Deutsche  med.  Wchnschr.,  (1885)  No.  19.  Gottstein, 
Fortschr.'d.  Med.,  (1885)  No.  16.  De  Michele  and  Radice,  Gior.  internets, 
di  sc.  Med.,  (1892)  535.  Sabouraud,  Ann.  de  iV/ist.  Pasteur,  vi.  184. 
Golasz,  Journ.  d.  mal.  cutan.  et  syph.,  (1894)  170.  Markuse,  Vrtljschr.f. 
Dermat.  u.  Syph.,  (1883)  No.  3.  van  Niessen,  Centralbl.  f.  Bakteriol.  u. 
Parasitenk.,  xxiii.  49.  Joseph  u.  Piorkowski,  Berl.  klin.  Woch.,  (1902)  No. 
13,  282. 

CHAPTER  X.  — TUBERCULOSIS. 

Klencke,  "  Untersuchungen  und  Erfuhrungen  im  Gebiet  der  Anatomic, 
etc.,"  Leipzig,  1843.  Villemin,  "  De  la  virulence  et  de  la  specificite  de  la 
tuberculose,"  Paris,  1868.  Cohnheim  u.  Fraenkel.  "  Experimentelle  Un- 
tersuchungen iiber  der  Ubertragbarkeit  der  Tuberculose  aufThiere."  Cohnheim, 
'•Die  Tuberculose  vom  Standpunkt  der  Infectionslehre,"  1879.  Various 
Authors,  "Discussion  sur  la  tuberculose,"  Bull.  Acad.  de  med.,  (1867)  xxxii., 
xxxiii.  Armanni,  "  Novimento  med.-chir."  Naples,  1872.  Baumgarten, 
"  Lehrb.  d.  path.  Myk.,"  1890.  Straus,  "La  tuberculose  et  son  bacille," 
Paris,  1895.  Koch,  Berl.  klin.  Wchnschr.,  (1882)  221;  Mitth.  a.  d.  k. 
Gsndhtsamte,  1884;  Deutsche  med.  Wchnschr.,  (1890)  No.  46  a;  (1891) 
Nos.  3  and  43;  (1897)  No.  14.  Bulloch,  Lancet,  (1901),  ii.  243.  Nocard, 
"The  Animal  Tuberculoses,"  (trans.)  London,  1895.  Cornet,  Ztschr.f.  Hyg., 
v.  191.  Nocard  et  Roux,  Ann.  de  Vlnst.  Pasteur,  i.  19.  Pawlowsky, 
ibid.  ii.  303.  Sander,  Arch.  f.  Hyg.,w\.  238.  Coppen  Jones,  Centralbl.  f. 
Bakteriol.  it.  Parasitenk.,  xvii.  i.  Prudden  and  Hodenpyl,  New  York  Med. 
Rec..  (1891)  636.  Vissman,  Vir chow's  Archiv,  cxxix.  163.  Straus  et 
Gamaleia,  Arch,  de  med.  exper.  et  (Tanat.  path.,  iii.  No.  4.  Courmont, 
Semaine  med.,  (1893)  53;  Revue  de  med.,  (1891)  No.  10.  He'ricourt  et 
Richet,  Bull,  med.,  (1892)  741,  966.  Williams,  Lancet,  (1883)  i.  312. 
Pawlowsky,  Ann.  de  FTnst.  Pasteur,  vi.  1 16.  Smith,  1\\..,Journ.  Exper.  Med., 
(1899)  iv.  217.  Maffucci,  "  Sull  azione  tossica  dei  prodotti  del  bacillo  della 
tuberculosi  "  ;  Centralbl  f.  attg.  Path.  u.  path.  Anat.,  i.  404.  Kruse,  Beitr. 
2.  path.  Anat.  u.  2.  attg.  Path.,  xii.  221 .  Bellinger,  Miinchen.  med.  Wchnschr., 
(1889)  No.  37;  Verhandl.  d.  Gesellsch.  deutsch.  Naturf.  u.  Aertze,  (1890) 
ii.  187.  Hofmann,  Wien.  med.  Wchnschr.,  (1894)  No.  38.  Straus  et  Wurtz, 
Cong.  p.  r etude  de  la  tuberculose,  Paris,  July,  1888.  Gilbert  et  Roger,  Mem. 
Sac.  de  biol.,  (1891).  Diem,  Monatsh.  f.  prakt.  Thierh.,  iii.  481.  Weyl, 
Deutsche  med.  Wchnschr.,  (1891)  256.  Buchner,  Centralbl.  f.  Bakteriol.  u. 
Parasitenk.,  xi.  488.  Courmont  et  Dor,  Province  med.,  (1890)  No.  50. 
Tizzoni  u.  Centanni,  Centralbl  f.  Bakteriol  u.  Parasitenk.,  xi.  82.  Ribbert, 
Deutsche  med.  Wchnschr.,  (1892)  353.  Virchow,  ibid.,  (1891)  131.  Hunter, 
Brit.  Med.  Journ.,  (1891)  July  25.  Kuhne,  Ztschr.  f.  Biol,  xxix.  i;  xxx. 
221.  Krehl,  Arch.  f.  exper.  Path.  u.  Pharmakol,  xxxv.  222.  Krehl  u. 
Matthes,  ibid.,  xxxvi.  437.  Bang,  "La  lutte  centre  la  tuberculose  en  Dane- 
mark,"  Geneva,  1895.  Maragliano,  "  Le  serum  antituberculeux  et  son  anti- 
toxin," Paris,  1896;  Berl  klin.  Wchnschr.,  (1896)  409,  437,773-  Nocard, 


542  BIBLIOGRAPHY. 

Ann,  de  rinst,  Pasteur,  xii.  561.  Stockman,  Brit.  Med.  Journ.,  (1898)  ii. 
601.  Maragliano,  ref.  Brit.  Med.  Journ.,  Epitome,  (1896)  i.  63.  Baumgarten 
u.  Walz,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  xxiii.  587.  Smith,  T., 
Journ.  Exper.  Med.,  iii.  451.  Koch,  Brit.  Med.  Journ.,  (1901)  ii.  189;  Trans- 
Inter  nat.  Congr.  of  Tuber c.,  London,  1901.  Repp,  Amer.  Med.,  (1901)  Oct. 
26,  Nov.  2.  Delepine,  Brit.  Med. Journ.,  (1901)  ii.  1224.  Bataillon,  Dubard,. 
et  Terre  (fish  tuberculosis),  Compt.  rend.  Soc.  de  biol.,  1897,  446.  Dubard^ 
Rev.  de  la  tuber  ciil.,  1898,  13,  129.  Ravenel,  Med.  Bull.  Univ.  Pennsylvania? 
May,  1892. 

Other  acid-fast  bacilli.  —  Moeller,  Deutsche  med.  IVchnschr.,  1898,  376. 
Centralbl.  f.  Bakteriol.  21.  Parasitenk.,  xxv.  369;  ibid.,  xxx.  513;  Deutsch. 
med.  Woch.,  (1902)  June  26,  466.  Petri,  Arb.  a.  d.  k.  Gsndhtsamte.,  1898,  I  ;. 
Hyg.  Rundsch.,  (1897)  vii.  811.  Rabinowitsch,  Deutsche  med.  Wchnschr., 
1897,  No.  26;  1900,  No.  16;  Lancet,  (1901)  Sept.  28,  838;  Ztschr.  f.  Hyg.r 
xxvi.  90.  Korn,  Arch.  f.  Hyg.,  xxxvi.  57  ;  Centralbl.  f.  Bakteriol.  u.  Para- 
sitenk., xxvii.  481.  Schulze,  Ztschr.  f.  Hyg.,  xxxi.  153.  M.  Tobler,  ibid.,. 
xxxvi.  1 20.  Lubarsch,  ibid.,  xxxi.  187.  Holscher,  Centralbl.  f.  Bakteriol.  u. 
Parasitenk.,  xxix.  425.  Potet,  "Etude  sur  les  bacilles  dites  <  acidophiles,' " 
Paris,  1902.  Arloing  et  Courmont,  Compt.  rend.  Acad.  des  Sc.,  (1898)  Aug.  8, 
Sept.  19.  Beck  u.  Rabinowitsch,  Deutsch.  med.  Woch.,  (1900)  No.  25,  400, 
(1901)  No.  10,  145.  R.  Koch,  ibid.,  (1901)  No.  48,  829. 

CHAPTER  XL  — LEPROSY. 

Hansen,  Norsk.  Mag.f.  Lagevidensk.,  1874 ;  Vir chow's  Archiv,  Ixxix.  32  ; 
xc.  542;  ciii.  388;  Virchow's  Festschr.,  (1892)  iii.  See  papers  by  Neisser 
and  Cornil  and  Suchard  in  "  Microparasites  in  Disease "  {New  Sydenham 
Soc.,  1886).  Hansen  and  Looft,  u  Leprosy,"  Bristol,  1895.  Doutrelepont 
u.  Wolters,  Arch.  f.  Dermat.  u.  Syph.,  (1892)  55.  Thoma,  Sitzungsb.  d* 
Dorpater  Naturforsch.,  1889.  Unna,  Dermat.  Stud.,  Hamburg,  (1887)  iv. 
Bordoni-Uffreduzzi,  Ztschr.  f.  Hyg.,  iii.  178;  Berl.  klin.  Wchnschr.,  (1885) 
No.  ii.  Arning  u.  Nonne,  Vir chow's  Archiv,  cxxxiv.  319.  Gairdner,. 
Brit.  Med.  Journ.,  (1887)  i.  1269.  Hutchinson,  Arch.  Surg.,  (1889)  i. 
V.  Torok,  "Summary  of  Discussion  on  Leprosy  at  the  1st  Internat.  Congr. 
for  Dermatol.  and  Syph.,"  v. ;  Monatsh.  f.  prakt.  Dermat.,  ix.  238.  Profeta,. 
Gior.  internaz.  d.  sc.  med.,  1889.  See  Journal  of  the  Leprosy  Investigation 
Committee,  1890-91.  Philippson,  Vir chow's  Archiv,  cxxxii.  529.  Danielssen,. 
Monatsh.  f.  prakt.  Dermat.,  (1891)  85,  142.  Wesener,  Centralbl.  f.  Bakteriol. 
u.  Parasitenk.,  ii.  450;  Munchen.  med.  Wchnschr.,  (1887)  No.  18.  Uhlen- 
huth  u.  Westphal,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  xxix.  233. 

CHAPTER  XII.  —  GLANDERS  —  RHINOSCLEROMA. 

Loffler  u.  Schiitz,  Deutsche  med.  Wchnschr.,  (1882)  No.  52.  Loftier,. 
Mitth.  a.  d.  k.  Gsndhtsamte.,  i.  134.  Weichselbaum,  Wien.  med.  Wchnschr. > 
(1885)  Nos.  21-24.  Preusse,  Berl.  thierdrztl.  Wchnschr.,  (1889)  Nos.  3,  5, 
ii;  ibid.,  (1894)  Nos.  39,  51.  Gamaleia,  Ann.  de  Vlnst.  Pasteur,  iv.  103. 
A.  Babes,  Arch,  de  med.  explr.  et  d'anat.  path.,  (1892)  450.  Straus,  Compt. 


BIBLIOGRAPHY.  543 

rend.  Acad.  d.  sc.,  cviii.,  530.  M'Fadyean  and  Woodhead,  Rep.  National 
Vet.  Assoc.,  1888.  Baumgarten,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  iii. 
397.  Silviera,  Semaine  med.,  (1891)  No.  31.  Bonome,  Deutsche  med. 
Wchnschr.,  (1894)  703,  725.  744.  Kalning,  Arch.  f.  Veterinarivissensch  , 
(St.  Petersburg)  i.  Apr.,  May.  Foth,  Centralbl.  f.  Bakteriol.  u.  Parasitenk., 
xvi.  508,  550.  M'Fadyean,  Journ.  Comp.  Path,  and  Therap.,  1892,  1893, 
1894.  Leclaiche  et  Montane",  Ann.  de  VInst.  Pasteur,  vii.  481.  Leo, 
Ztschr.  f.  Hyg.,  vii.  505.  Marx,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  xxv. 
275.  Mayer,  ibid.,  xviii.  673.  Frothingham,  Journ.  Med.  Research,  (1901) 
n.s.  i.  No.  2,  331. 

RHINOSCLEROMA.  —  Frisch,  Wien.  med.  Wchnschr.,  (1882)  No.  32. 
Cornil  et  Alvarez,  Arch,  de  physiol.  norm,  et  path.,  1895,  3rd  series,  vi.  11. 
Paltauf  u.  Eiselsberg,  Fortschr.  d.  Med.,  (1886)  Nos.  19,  20.  Wolko- 
witsch,  Centralbl.  f.  d.  med.  Wissensch.,  1886.  Dittrich,  Ztschr.  f.  Heilk., 
viii.  251.  Babes,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  ii.  617.  Pawlowski, 
ibid.,  ix.,  742 ;  "  Sur  Pe'tiologie  et  la  pathologic  du  rhinosclerome,"  Berlin, 
1891.  Paltauf,  Wien.  med.  Wchnschr.,  (1891)  Nos.  52,  53;  (1892)  Nos.  i,  2. 
Wilde,  Semaine  med.,  (1896)  336. 


CHAPTER  XIIL  — ACTINOMYCOSIS,  ETC. 

Bellinger,  Centralbl.  f.  d.  med.  Wissensch.,  1877.  J.  Israel,  Vir chow's 
Archiv,  Ixxiv.  15;  Ixxviii.  421.  Ponfick,  Breslau.  aertzl.  Ztschr.,  1879; 
"Die  Aktinomykose  des  Menschen,"  1882.  O.  Israel,  Virchow's  Archiv, 
xcvi.  175.  Chiari,  Prag.  med.  Wchnschr.,  1884.  Langhans,  Cor.-Bl.  f. 
schweiz.  Aerzte,  xviii.  (1888).  Liming  u.  Hanau,  ibid.,  xix.  (1889). 
Shattock,  Trans.  Path.  Soc.  London,  1885.  Acland,  ibid.,  1886.  Delepine, 
ibid..  1889.  Harley,  Med.-Chir.  Trans.,  London,  1886.  Crookshank,  ibid., 
1889;  "Manual  of  Bacteriology,"  London,  1896.  Ransome,  Med.-Chir. 
Trans.,  London,  1891.  M'Fadyean,  Journ.  Comp.  Path,  and  Therap.,  1889. 
Bostrbm,  Beitr.  2.  path.  Anat.  u.  2.  allg.  Path.,  1890.  Wolff  u.  Israel, 
Virchow*s  Archiv,  cxxvi.  u.  Illich,  "Beitrage  zur  Klinik  der  Aktinomykose,1' 
Wien,  1892.  Grainger  Stewart  and  Muir,  Edin.  Hos.  Rep.,  1893.  Leith, 
ibid.,  1894.  Gasperini,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  xv.  684. 
Hummel,  Beitr.  2.  klin.  Chir.,  xiii.  No.  3.  Pawlowsky  et  Maksutoff,  Ann. 
de  r Inst.  Pasteur,  vii.  544. 

Allied  Streptothrices.  —  ^wd,t&,  Ann.  de  rinst.  Pasteur,  ii.  1888,  293. 
Eppinger,  Beitr.  2.  path.  Anat.  u.  z.  allg.  Path.,  ix,  287,  "  Ergebnisse  der 
Allgem.  Path."  (Lubarsch  u.  Ostertag),  iii.  328.  Buchholz,  Ztschr.  f. 
Hyg.,  xxiv,  470.  Berestnew,  ibid.,  xxix.  94.  Flexner,/<?w«.  Exper.  Med.,  iii. 
435.  Dean,  Trans.  Path.  Soc.  London,  1900,  26.  Birt  and  Leishman, /<?«/-«. 
of  Hyg.,  ii.  120.  Mertens,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  xxix.  649. 
Foulerton,  Trans.  Path.  Soc.  London,  1902,  56. 

MADURA  DISEASE.  —  Carter,  "  On  Mycetoma  or  the  Fungus  Disease  of 
India,"  London.  Bassini,  Ref.  in  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  iv. 
652.  Lewis  and  Cunningham,  nth  Ann.  Rep.  San.  Com.  India.  Kobner, 
Fortschr.  d.  Med.,  (1886)  No.  17.  Kanthack,  Journ.  Path,  and  Bacteriol., 
i.  140.  Boyce  and  Surveyor,  Proc.  Roy.  Soc.  London,  1893.  Vandyke  Carter, 


544  BIBLIOGRAPHY. 

Trans.  Path.  Soc.  London,  1886.     Vincent,  Ann.  de  rinst.  Pasteur,  viii.  129. 
Wright,  J.  H.,Journ.  Exper.  Med.,  iii.  421. 

CHAPTER  XIV.  —  ANTHRAX. 

Bellinger  in  Ziemssen's  "  Cyclopaedia  of  Medicine."  Greenfield,  "  Malig- 
nant Pustule  "  in  Quain1s  "  Dictionary  of  Medicine,11  London,  1  894.  Pollender, 
Vrtljschr.  f.  gerichtl.  Med.,  viii.  ;  Davaine,  Compt.  rend.  Acad.  d.  sc.,  Ivii.  220, 
351,  386;  lix.  393.  Koch,  Cohn's  Beitr.  -z.  Biol.  d.  Pflanz.,  ii.  Heft  2  (1876). 
Mitth.  a.  d.  k.  Gsndhtsamte.,  i.  49.  Pasteur,  Compt.  rend.  Acad.  d.  sc.,  xci.  86, 
455'  53  J>  697  ;  xcii.  209.  Buchner,  Virchow's  Archiv,  xci.  Chamberland,  Ann. 
de  rinst.  Pasteur,  viii.  161.  Chauveau,  Compt.  rend.  Acad.  d.  sc.,  xci.  33, 
648,  880;  xcvi.  553.  Czaplewski,  Beitr.  2.  path.  Anat.  u.  2.  allg.  Path.,  vii. 
47.  Gamaleia,  Ann.  de  rinst.  Pasteur,  ii.  517.  Marshall  Ward,  Proc.  Roy. 
Soc.  London,  Feb.  1893.  Petruschky,  Beitr.  z.  path.  Anat.  u.  z.  allg.  Path., 
iii.  357.  Weyl,  Ztschr.  f.  Hyg.,  xi.  381.  Behring,  ibid.,  vi.  117:  vii.  171. 
Osborne,  Arch.  f.  Hyg.,  xi.  51.  Roux,  Ann.  de  Vlnsl.  Pasteur,  iv.  25. 
Hankin,  Brit.  Med.  Journ.,  (1889)  ii.  810;  (1890)  ii.  65.  Hankin  et  Wes- 
brook,  Ann.  de  rinst.  Pasteur,  vi.  633.  Sidney  Martin,  Rep.  Med.  Off.  Loc. 
Govt.  Board,  (1890-91)  255.  Marmier,  Ann.  de  rinst.  Pasteur,  ix.  533.  Rd. 
;j.  Path,  and  Bacterial.,  v.  374. 


CHAPTER  XV.—  TYPHOID  FEVER,  ETC. 

Eberth,  Virchoitfs  Archiv,  Ixxxi.  58  ;  Ixxxiii.  486.  Koch,  Mitth.  a.  d.  k. 
Gsndhtsamte.,  \.  46.  Gaffky.  ibid.,  ii.  80.  Klebs,  Arch.  f.  exper.  Path.  u. 
Pharmakol.,  xii.  231  ;  xiii.  381.  Escherich,  Fortschr.  d.  Med.,  (1885)  Nos. 
1  6,  17.  Emmerich,  Arch.  f.  Hyg.,  iii.  291.  Rodet  et  Roux,  Arch,  de  med. 
exper.  et  d^anat.  path.,  iv.  317.  Weisser,  Ztschr.  f.  Hyg.,  i.  315.  Klein, 
"Micro-organisms  and  Disease,11  London,  1896;  Rep.  Med.  Off.  Loc.  Govt. 
Board,  (1892-93)  345;  (1893-94)  457;  (1894-95)  399,407,411.  Babes, 
Ztschr.  f.  Hyg.,  ix.  323.  Vincent,  Compt.  rend.  Soc.  de  biol.,  ser.  ix.  ii.  62. 
Birch-Hirschfeld,  Arch.  f.  Hyg.,  vii.  341.  Buchner,  Centralbl.  f.  Bakteriol.  u. 
Parasitenk.,  iv.  353.  Pfuhl,  ibid.,  iv.  769.  Petruschky,  ibid.,  vi.  660.  Dur- 
ham, Jouru.  Exper.  Med.,  (1901)  v.  353.  Hunter,  Lancet,  (1901)  i.  613. 
Kitasato,  Ztschr.  f.  Hyg.,  vii.  515.  Chantemesse  et  Widal,  Bull,  med.,  (1891) 
No.  82;  Ann.  de  rinst.  Pasteur,  vi.  755;  vii.  141.  Pere,  Ann.  de  rinst. 
Pasteur,  vi.  512.  Neisser,  Ztschr.  f.  klin.  Med.,  xxiii.  93.  Nicholle,  Ann.  de 
rinst.  Pasteur,  viii.  853.  Quincke  u.  Stuhlen,  Berl.  klin.  Wchnschr.,  (1894) 
351.  A.  Fraenkel,  Centralbl.  f.  klin.  Med  .  (1886)  No.  10.  E.  Fraenkel  u. 
Simmonds,  ibid.,  (1886)  No.  39.  Achalme,  Semaine  med.,  (1890)  No.  27. 
Grawitz,  Charity-Ann.,  xvii.  228.  Beumer  u.  Peiper,  'Centralbl.  f.  klin.  Med., 
(1887)  No.  4;  Zi.chr.  f.  Hyg.,  i.  489;  ii.  no,  382.  Sirotinin,  ibid.,  i.  465. 
R.  Pfeiffer  u.  Kolle,  Ztschr.  f.  Hyg.,  xxi.  203.  R.  Pfeiffer,  Deutsche  med. 
Wchnschr.,  (1894)  898.  Sanarelli,  Ann.  de  rinst.  Pasteur,  vi.  721  ;  viii.  193, 
353.  Brieger  u.  Fraenkel,  Berl.  klin.  Wchnschr.,  (1890)  241,  268.  Brieger, 
Kitasato,  u.  Wassermann,  Ztschr.  f.  Hyg.,  xii.  137.  Widal,  Semaine  7ned., 
(1896)  295,  303.  Achard,  ibid.,  295,  303.  Griinbaum,  Lancet,  Sept.  1896. 


BIBLIOGRAPHY. 


545 


Delepine,  Brit.  Med.  Journ.,  (1897)  i.  529, 967  ;  Lancet,  Dec.  1896.  Remlinger 
et  Schneider,  Ann.  de  rinst.  Pasteur,  xi.  55,  829.  Widal  et  Sicard,'#/V/., 
xi.  353.  Peckham,  Journ.  Exper.  Med.,  ii.  549.  Richardson,  ibid.,  iii.  349! 
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28.  Wright  and  Semple,  Brit.  Med.  Journ.,  (1897)  i.  256.  Wright  and 
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Exper.  Med.,  (1897)  ii.  677  ;  Journ.  Med.  Research,  (1902)  June,  148.  Piorkow- 
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(1897)  ii.  1746.  Durham,  Lancet,  (1898)  i.  154;  ibid.,  ii.  446.  Lorrain 
Smith  and  Tennant,  Brit.  Med.  Journ.,  (1899)  i.  193.  Gordon,  Jouru.  Path. 
and  Bacteriol.,  iv.  438.  Widal  et  Nobe'court,  La  Semaine  med.,  (1897)  285. 
Gwyn,  Johns  Hopkins  Hosp.  Bull.,  (1898)  ix.  54.  Schottmiiller,  Deut.  med. 
Woch.,  (1900)  No.  32,  511;  Zeit.  f.  Hyg.  u.  Infektskr.,  (1901)  xxxvi.  368. 
Johnston,  Amer.  Journ.  med.  Sc.,  (1902)  Aug.  Longcope,  ibid.  Hewlett, 
ibid.  Buxton,y<??/r;/.  med.  Research,  (1902)  June,  201.  (Bacillus  enteritidis, 
Gaertner),  refs.  vide  Bauingarten's  Jahrcsbericht,  iv.  249;  vii.  297;  xii.  508. 
(Psittacosis),  ibid.,  xii.  496.  Chiari  u.  Kraus,  Zeit.  f.  Heilkunde,  (1897)  Heft 
5  and  6,471.  Flexner  and  Harris,  Johns  Hopkins  Hosp.  Butt.,  (1897)  No. 
8 1 .  Lartigau,  New  York  Med.  Journ.,  (1899)  Jan.  29 ;  Johns  Hopkins  Hosp. 
Bull.,  (1899)  April.  Ophiils,  New  York  Med.  Journ.,  (1900)  May  12.  Opie 
and  Bassett,  Johns  Hopkins  Hosp.  Bull.,  (1901)  July,  198.  (Bacillus  enteriti- 
dis sporogenes),  Klein,  Rep.  Med.  Off.  Local  Govt.  Board,  xxv.  171  ;  xxvii.  210. 
BACILLARY  DYSENTERY.  —  Shiga,  Centralbl.  f.  Bakteriol.  u.  Parasitenk., 
xxiii.  599;  xxiv.  817,  870,  913;  Deutsch.  med.  Woch.,  (1901)  Nos.  43, 
44,45.  Kruse,  Deutsche  med.  Wchnschr.,  (1900)  637.  Flexner,  Bull.  Johns 
Hopkins  Hosp.,  (1900)  xi.  39,  231  ;  Brit.  Med.  Journ.,  (1900)  ii.  917; 
Philadel.  Med.  Journ.,  (1900)  vi.  414;  Uni-v.  Penn.  Med.  Bull.,  (1901)  No.  6, 
190.  Strong  and  Musgrave,  Journ.  Amer.  Med.  Assoc.,  (1900)  xxxv.  498. 
Vedder  and  Duval,/0»r«.  Exper.  Med.,  (1902)  vi.  181.  Duval  and  Bassett, 
Amer.  Med.,  (1902)  iv.  No.  ii,  417.  Spronck,  Nederl.  Tijdschft.  v. 
Geneesknde.,  (1902)  ii.  No.  18.  Ogata,  Centralbl.  f.  Bakteriol.  u.  Parasitenk., 
xi.  264. 

CHAPTER  XVI.— DIPHTHERIA. 

Klebs,  Verhandl.  d.  Cong.  f.  inner e  Med.,  (1883)  ii.     Loffler,  Mitth.  a.  d. 
k.   Gsndhtsamte.,  (1884)  421.     Roux  et  Yersin,  Ann.  de  VInst.  Pasteur,  ii. 


546  BIBLIOGRAPHY. 

629;  iii.  273;  iv.  385.  Brieger  u.  Fraenkel,  Berl.  klin.  Wchnschr.,  (1890) 
241,  268.  Spronck,  CentralbL  f.  allg.  Path.  u.  path.  Anat.,  i.  No.  25  ;  iii. 
No.  i.  Welch  and  Abbott,  John  Hopkins  Hasp.  Bull,  1891.  Councilman, 
Mallory,  and  Pearce,  Journ.  Boston  Soc.  Med.  Sc.,  (1900)  Dec.  Behring  u. 
Wernicke,  Ztschr.  f.  Hyg.,  xii.  10.  Lbffler,  CentralbL  f.  Bakteriol.  u.  Parasi- 
tenk., ii.  105.  v.  Hofmann,  Wien.  med.  Wchnschr.,  (1888)  Nos.  3  and  4. 
Wesbrook,  Wilson,  and  McDaniel,  g7rtf/z.y.  Assoc.  Amer.  Phys.,  (1900)  xv. 
198.  Hill,  Journ.  Med.  Research,  (1902)  ii.  N.S.  115;  Ann.  Rep.  Boston 
Board  of  Health,  1901.  Cobbett  and  Phillips.  Jo /trn.  Path,  and  Bacterial.,  iv. 
193.  Peters,  ibid.,  iv.  181.  Wright,  Boston  Med.  and  S.  Journ.,  (1894)  329, 
357.  Kanthack  and  Stephens,  Joum.  Path,  and  BacterioL,  iv.  45.  Klein, 
Brit.  Med.  Journ.,  (1894)  ii.  1393;  (1895)  i.  100.  Rep.  Med.  Off.  Loc. 
Govt.  Board,  (1890-91)  219;  (1891-92)  125.  Abbott,  Journ.  Path,  and 
Bact.,  (1893)  Oct.  Guinochet,  Compt.  rend.  Soc.  de  biol.,  (1892)  480.  Roux 
et  Martin,  Ann.  de  Plnst.  Pasteur,  viii.  609.  Cartvvright  Wood,  Lancet, 
(1896)  i.  980,  1076;  ii.  1145.  Sidney  Martin,  "  Goulstonian  Lectures,11 
Brit.  Med.  Journ.,  (1892)  i.  641,  696,  755;  Rep.  Med.  Off.  Loc.  Govt. 
Board,  (1891-92)  147;  (1892-93)  427.  Escherich,  Wien.  med.  Wchnschr., 
(1893)  Nos.  47-50;  Wien.  klin.  Wchnschr.,  (1893)  Nos.  7-10;  (1894) 
No.  22;  Berl.  klin.  Wchnschr.,  (1893)  Nos.  21,  22,  23.  Behring,  "Die 
Geschichte  der  Diphtheric,"  Leipzig,  1893;  "  Abhandlungen  z.  atiol.  Therap. 
v.  anst.  Krankh.,"  Leipzig,  1893;  "  Bekampfung  der  Infections-krankheiten," 
Leipzig,  1894.  Ehrlich  u.  Wassermann,  Ztschr.  f.  Hyg.,  xviii.  239.  Ehrlich 
u.  Kossel,  ibid.,  xvii.  486.  Ehrlich,  Kossel,  u.  Wassermann,  Deutsche 
med.  Wchnschr.,  (1894)  353.  Funck,  Ztschr.  f.  Hyg.,  xvii.  401.  Prochaska, 
Ztschr.  f.  Hyg.,  xxiv.  373.  Madsen,  ibid.,  xxiv.  425.  Richmond  and  Salter, 
Guy's  Hosp.  Repts.,  vol.  53,  p.  55.  Lesieur,  Journ.  Physiolog.  et  Path.  Gene- 
ral, (1901)  iii.  961,  1000.  Neisser,  Ztschr.  f.  Hyg.,  xxiv.  443.  L.  Martin, 
Ann.  de  1'Tnst.  Pasteur,  xii.  26.  Salomonsen  et  Madsen,  ibid.,  xii.  763. 
Woodhead,  Brit.  Med.  Journ.,  (1898)  ii.  893;  Rep.  Metrop.  Asyl.  Bd., 
London,  1901.  Me"tin,  Ann.  de  VInst.  Pasteur,  xii.  596.  Madsen,  ibid.,  xiii. 
568,  801.  Dean  and  Todd,  Journ.  of  Hyg.,  ii.  194.  Gobbett,  ibid.,  i.  485. 

CHAPTER   XVII.  — TETANUS,  ETC. 

Nicolaier,  "  Beitrage  zur  Aetiologie  des  Wundstarrkrampfes,"  Inaug.  Diss. 
Gottingen,  1885.  Rosenbach,  Arch.  f.  klin.  Chir.,  xxxiv.  306.  Carle  and 
Rattone,  Gior.  d.  r.  Accad.  di  med.  di  Torino,  1884.  Kitasato,  Ztschr.  f. 
Hyg.,  vii.  225;  x.  267;  xii.  256.  Kitasato  u.  Weyl,  ibid.,  viii.  41,  404. 
Vaillard,  Ann.  de  rinst.  Pasteur,  vi.  224,  676.  Vaillard  et  Rouget,  ibid., 
vi.  385.  Behring,  "Abhandlungen  z.  atiol.  Therap.  v.  anst.  Krankh.," 
Leipzig,  1893;  Ztschr.  f.  Hyg.,  xii.  i,  45;  "  Blutserumtherapie,"  Leipzig, 
1892;  "Das  Tetanusheilserum,11  Leipzig,  1892.  'Brieger  u.  Fraenkel, 
Berl.  klin.  Wchnschr.,  (1890)  241,  268.  Sidney  Martin,  Rep.  Med.  Of. 
Loc.  Gout.  Board,  (1893-94)  497;  (1894-95)  505.  Uschinsky,  Centralbl.  f. 
Bakteriol.  u.  Parasitenk.,  xiv.  316.  Bolton  and  Fisch,  Trans.  Assoc.  Amer. 
Phys.,  (1902)  xvii.  462.  Tizzoni  u.  Cattani,  Arch.  f.  exper.  Path.  u.  Phar- 
makol.,  xxvii.  432;  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  ix.  189,  685;  x. 


BIBLIOGRAPHY.  547 


33,  576  (Ref.)  ;  xi.  325  ;  Berl  klin.  Wchnschr.,  (1894)  732.     Madsen, 

/.  Hyg.,  xxxii.  214.     Ritchie,  Jonrn.  of  Hyg..  i.  125.     Danysz,  Ann.  de  Vlnst. 

Pasteur,  xiii.  155. 

MALIGNANT  (EDEMA.  —  Pasteur,  Bull.  Acad.  de  mid.,  1  88  1,  1887.  Koch, 
Mitth.  a.  d.  k.  Gsndhtsamte.,  i.  54.  Kitt,  Jahresb.  d.  k.  Centr.-Thierarznei- 
Schule  in  Munchen,  1883-84.  W.  R%  Hesse,  Deutsche  med.  Wchnschr., 
(1885)  No.  14.  Chauveau  et  Arloing,  Arch,  vet.,  (1884)  366,  817.  Libo- 
rius,  Ztschr.f.  Hyg.,  i.  115.  Roux  et  Chamberland,  Ann.  de  Vlnst.  Pasteur, 
\.  562.  Charrin  et  Roger,  Compt.  rend.  Soc.  de  biol.,  (1877),  s^r-  vin.  iv. 
408.  Kerry  u.  S.  Fraenkel,  Ztschr.  f.  Hyg.,  xii.  204.  Sanfelice,  ibid.,  xiv. 
339.  Leclainche  et  Velle,  Ann.  de  I1  hist.  Pasteur,  xiv.  202,  590. 

BACILLUS  BOTULINUS.  —  V.  Ermengem,  Centralbl.f.  Bakteriol.  u.  Parasi- 
tenk.,  xix.  443  ;  Ztschr.f.  Hyg.,  xxvi.  i.  Kempner,  ibid.,  xxvi.  481.  Kemp- 
ner  u.  Schepilewsky,  ibid.,  xxvii.  213.  Kempner  u.  Pollack,  Deutsche  med. 
Wchnschr.,  1897,  No.  32.  Brieger  u.  Kempner,  ibid.,  1897,  No.  33.  Mari- 
nesco,  Compt.  rend.  Soc.  de  Biol.,  1896,  No.  31.  Schneidemiihl,  Centralbl.f. 
Bakteriol.  u.  Parasitenk.,  xxiv.  577,  619.  Romer,  ibid.,  xxvii.  857. 

QUARTER-EVIL.  —  See  Nocard  et  Leclainche,  "Les  maladies  micro- 
biennes  des  animaux,"  Paris  (1896).  Arloing,  Come  vin,  et  Thomas,  uLe 
charbon  symptomatique  du  boeuf,"  Paris  (1887).  Nocard  et  Roux,  Ann.  de 
Vlnst.  Pasteur,  i.  256.  Roux,  ibid.,  ii.  49.  See  ulsojourn.  Comp.  Path,  and 
Therap.,  iii.  253,  346;  viii.  166,  233. 

BACILLUS  AEROGENES  CAPSULATUS.  —  Welch  and  Nuttall,  Bull.  Johns 
Hopkins  Hasp.,  1892,  81.  Welch  and  Flexner,  Journ.  Exper.  Med.,  i.  5. 
E.  Fraenkel,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  xiii.  13.  E.  Fraenkel, 
Ueber  Gasphlegmonen,  (Hamburg  u.  Leipzig,  1893).  Dunham,  Bull.  Johns 
Hopkins  Hosp.,  1897,  No.  73,  68.  Norris,  Am.  Journ.  Med.  Sc.,  cxvii.  172. 
Howard,  Contrib.  to  the  Sc.  of  Med.,  dedicated  by  his  pupils  to  William  H. 
Welch,  Baltimore,  (1900)  461.  Bloodgood,  Progress.  Med.,  (1899)  iv.  158. 
Fulton  and  Pratt,  Boston  Med.  and  Surg.  Journ.,  (1900)  599.  Welch,  Johns 
Hopkins  Hosp.  Bull.,  (1900)  xi.  185;  Centralbl.  f.  Bakt.  u.  Parasitenk., 
Abt.  i.,  (1901)  Bd.  xxix.  442.  Gvvyn,  Johns  Hopkins  Hosp.  Bull.,  (1899) 
x.  134.  Cole,  ibid.,  (1902)  xiii.  234. 

CHAPTER  XVIII.  —  CHOLERA. 

Koch,  Rept.  of  \st  Cholera  Conference,  1884  (v.  "  Microparasites  in  Dis- 
ease," New  Sydenham  Soc.,  1886).  Nikati  et  Rietsch,  Compt.  rend.  Acad. 
d.  sc.,  xcix.  928,  1145.  Bosk,  Ann.  de  rinst.  Pasteur,  ix.  507.  Pettenkofer, 
Munchen.  ,  med.  Wchnschr.,  (1892)  No.  46;  (1894)  No.  10.  Sawtschenko, 
Centralbl.  f.  Bakteriol.  u.  Parasilenk.,  xii.  893.  Pfeiffer,  Ztschr.  f.  Hyg.,  xi. 
393.  Kolle,  ibid.,  xvi.  329.  Issaeff  u.  Kolle,  ibid.,  xviii.  17.  Wassermann, 
ibid.,  xiv.  35.  Sobernheim,  ibid.,  xiv.  485.  Metchnikoff,  Ann.  de  rinst. 
Pasteur,  vii.  403,  562;  viii.  257,  529.  Fraenkel  u.  Sobernheim,  Hyg. 
Rundschau,  iv.  97.  Dunbar,  Arb.  a.  d.  k.  Gsndhtsamte.,  ix.  379.  Pfeiffer 
u.  Wassermann,  Ztschr.  f.  Hyg.,  xiv.  46.  Wesbrook,  Ann.  de  rinst.  Pasteur, 
viii.  318.  Scholl,  Berl.  klin.  Wchnschr.,  (1890)  No.  41.  Griiber  u,  Wiener, 
Arch.  f.  Hyg.,  xv.  241.  Cunningham,  Scient.  mem.  med.  off.  India,  1890  and 


548  BIBLIOGRAPHY. 

1894.  Hueppe,  Deutsche  med.  WcJfnschr.,  (1889)  No.  33.  Klemperer,  ibid., 
(1894)  435  ;  Berl.  klin.  Wchnschr.,  (1892)  969.  Lazarus,  ibid.,  (1892)  1071. 
Reincke,  Deutsche  med.  Wchnschr.,  (1894)  795.  Koch,  Ztschr.  /.  Hyg.,  xiv. 
319.  Voges,  CentralbL  /.  Bakteriol.  n.  Parasitenk.,  xv.  453.  Pastana  u. 
Bettencourt,  CentralbL  f.  Bakteriol.  u.  Parasitenk.,  xvi.  401.  Dieudonne, 
ibid.,  xiv.,  323.  Celli  u.  Santori,  ibid.,  xv.  289.  Neisser,  ibid.,  xiv.,  666. 
Sanarelli,  Ann.  de  rinst.  Pasteur,  vii.  693.  Ivanoff,  Ztschr.  f.  Hyg.,  xv.  485. 
Issaefif,  ibid.,  xvi.  286.  Pfuhl,  ibid.,  x.  5 10.  Rumpel,  Deutsche  med.  Wchnschr., 
(1893),  1 60.  Klein.,  Rep.  Med.  Off.  Loc.  G(rut.  Board,  1893;  "Micro-organ- 
isms and  Disease,"  London,  1896.  Haffkine,  Brit.  Med.  Journ.,  (1895)  ii. 
1541.  Indian  Med.  Gaz.,  1895,  No.  I  ;  "  Anti-cholera  Inoculation,1'  Rep.  San. 
Com.  India,  Calcutta,  1895.  Pfeiffer  in  Flugge,  "Die  Mikroorganismen/1 
3rd  ed.,  1896 ;  Gamaleia,  Ann.  de  VInst.  Pasteur,  ii.  482,  552.  Abbott,  Journ. 
Exper.  Med.,  (1896)  i.  419.  Achard  et  Bensande,  Semaine  med.,  (1897) 
151.  Rumpf,  "  Die  Cholera  Asiatica  und  Nostras,"  Jena,  1898. 

CHAPTER  XIX.  — INFLUENZA,  ETC. 

INFLUENZA.  —  Pfeiffer,  Kitasato,  u.  Canon,  Deutsche  med.  Wchnschr., 
xviii.  28,  and  Brit.  Med.  Journ.,  (1892)  i.  128.  Babes,  Deutsche  med. 
Wchnschr.,  xviii.  113.  Pfeiffer  u.  Beck,  ibid.,  (1892)  465.  Pfuhl,  CentralbL 
f.  Bakteriol.  u.  Parasitenk.,  xi.  397.  Klein,  Rep.  Med.  Off.  Loc.  Govt.  Board, 

1 i 893)  85 •     Pfeiffer,  Ztschr.  f.  Hyg.,  xiii.  357.    Huber,  Ztschr.  f.  Hyg.,  xv.  454. 
Kruse,  Deutsche  med.  Wchnschr.,  (1894)  513.     Pelicke,  Berl.  klin.  Wchnschr., 

(1894)  524.      Pfuhl  u.  Walter,  Deutsche  med.    Wchnschr.,  (1896)  82,   105. 
Cantani,  Ztschr.  f.  Hyg.,  xxiii.  265.     Pfuhl,  Ztschr.  f.  Hyg.,  xxvi.  112.     Was- 
sermann,  Deutsche  med.  Wchnschr.,  1900,  No.  28.     Clemens,  Munchen.  med. 
Wchnschr.,  1900,  No.  27. 

PLAGUE.  —  Kitasato,  Lancet,  (1894)  ii.  428.  Yersin,  Ann.  de  rinst. 
Pasteur,  viii.  662.  Lowson,  Lancet,  (1895)  ii.  199.  Yersin,  Calmette,  et 
Borrel,  Ann.  de  rinst.  Pasteur,  ix.  589.  Aoyama,  CentralbL  f.  Bakteriol.  u. 
Parasitenk.,  xix.  481.  Zettnow,  Ztschr.  f.  Hyg.,  xxi.  164.  Yersin,  Ann.  de 
rinst.  Pasteur,  xi.  81.  Gordon,  Lancet,  (1899)  i.  688.  Wilson,  Journ.  Med. 
Research,  (1901)  i.,  n.  s.,  i.  53.  Haffkine,  Brit.  Med.  Journ.,  (1897)  i.  424. 
Wyssokowitz  et  Zabolotny,  Ann.  de  rinst.  Pasteur,  xi.  663.  Ogata,  Cen- 
tralbL f.  Bakteriol.  u.  Parasitenk.,  xxi.  769.  Childe,  Brit.  Med.  Journ.,  (1898) 
ii.  858.  See  also  Brit.  Med.  Journ.  and  Lancet,  1897-99.  Lustigu.  Galeotti, 
Deutsche  med.  Wchnschr.,  1897,  No.  15.  Markl,  CentralbL  f.  Bakteriol.  u. 
Parasitenk..  xxiv.  641,  728  ;  xxix.  810.  Cairns,  Lancet,  (1901)  i.  1746.  Mon- 
tenegro, "  Bubonic  Plague,"1  London,  1900.  Netter,  "  La  peste  et  son  bacille," 
Paris,  1900.  Mitth.  der  Deutschen  Pest-Kommission,  Deutsche  med.  Wchnschr., 
1897,  Nos.  17,  19,  31,  32.  Report  of  the  Indian  Plague  Commission  (1898- 
99),  London,  1900-01,  Also  numerous  papers  in  the  Lancet  and  Brit.  Med. 
Journ.,  1897-1901.  Regarding  Glasgow  epidemic  see  ibid.,  1900,  ii.  Barker, 
Am.  Journ.  Med.  Sc.,  (1901)  Oct.  Flexner,  ibid.  Novy,  ibid. 

RELAPSING  FEVER.  —  Obermeier,  CentralbL  f.  d.  med.  Wissensch.,  (1873) 
145  ;  andZter/.  klin.  Wchnschr.,  (1873)  No.  35.  MUnch,  CentralbL  f.  d.  med. 
Wissensch.,  1876.  Koch,  Deutsche  med.  Wchnschr.,  (1879)  327-  Moczut- 


BIBLIOGRAPHY.  549 

kowsky,  Deutsches  Arch.  f.  klin.  Med.,  xxiv.  192.  Vandyke  Carter,  Med.- 
Chir.  Trans.,  London,  (1880)  78.  Lubinoff,  Virchoitfs  Archiv,  xcviii.  160. 
Metchnikoff,  ibid.,  cix.  176.  Soudakewitch,  Ann.  de  rinst.  Pasteur,  v.  545. 
.Lamb,  Scient.  mem.  med.  off.  India,  1901,  pt.  xii.  77.  Sawtschenko  et 
Melkich,  Ann.  de  rinst.  Pasteur ^  xv.  497. 

MALTA  FEVER.  —  Bruce,  Practitioner,  xxxix.  160;  xl.  241;  Ann.  de- 
rinst.  Pasteur,  vii.  291.  Bruce,  Hughes,  and  Westcott,  Brit.  Med.  Journ.,. 
(1887)  ii.  58.  Hughes,  Ann.  de  rinst.  Pasteur,  vii.  628;  Lancet,  (1892)  ii 
1265.  Wright  and  Semple,  Brit.  Med.  Journ.,  (1897)  i.  1214.  Wright  and1: 
Smith,  ibid.,  (1897)  i.  911;  Lancet,  (1897)  i.  656.  Welch,  ibid.,  (1897)  i.. 
1512.  Gordon,  ibid.,  (1899)  i-  688-  Durham,  Journ.  Path,  and  Bacteriol.r 
v.  377.  Bruce  in  Davidson's  <*  Hygiene  and  Diseases  of  Warm  Climates," 
Edinburgh  and  London,  1893.  Birt  and  Lamb,  Lancet,  1899,  n<-  7O1-  Brun- 
ner,  Wiener  klin.  Wchnschr.,  1900,  No.  7.  Cox,  Philadel.  Med.  Journ.,  (1899) 
Sept.  9,  491.  Musser  and  Sailer,  Philadel.  Med.  Journ.,  (1898)  Dec.  31,  1408. 
Strong  and  Musgrave,  ibid.,  (1900)  Nov.  24.  Curry,  Journ.  Med.  Research^ 

(1901)  i.,  n.  s.,  i.  241. 

YELLOW  FEVER.  —  Sternberg,  Rep.  Am.  Pub.  Health  Ass.,  xv.  170.. 
Sanarelli,  Ann.  de  rinst.  Pasteitr,  xi.  433,  673,  753  ;  xii.  348.  Davidson,  art, 
in  Clifford  Allbutfs  "  System  of  Medicine,"  vol.  ii.,  London,  1897.  Sternbergr 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  xxii.  145  ;  xxiii.  769.  Sanarelli,  ibid.r 
xxii.  668.  Archinard,  Woodson,  ibid.,  xxv.  393,  (and  Archinard  ref.).  Archi- 
nard  and  Woodson,  New  York  Med.  Journ.,  (1899)  Jan.  28.  Mendoza  (ref.), 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  xxv.  390.  Wasdin  and  Geddings,  Re- 
port of  Treasury  Dept.,  Washington,  1899.  Reed,  Journ.  of  Hyg.,  ii.  101 
(with  full  references).  Reed  and  Carroll,  Journ.  Exper.  Med.,  (1900)  v.  215  ; 
Am.  Med.,  (1902)  Feb.  22,  301.  Durham,  Thompson-Yates  Laboratory  Rep.+ 

(1902)  iv.  pt.  ii.  485. 

CHAPTER  XX.  — IMMUNITY. 

For  early  inoculation  methods  {e.g.  against  anthrax,  chicken  cholera,, 
etc.),  see  "  Microparasites  in  Disease,"  New  Syd.  Soc.,  1886.  Duguid  and 
Sanderson,  Journ.  Roy.  Agric.  Soc.,  (1880)  267.  Greenfield,  ibid.,  (1880) 
273;  Proc.  Roy.  Soc.  London,  June,  1880.  Toussaint,  Compt.  rend.  Acad. 
d.  sc.,  xci.  135.  HafFkine,  Brit.  Med.  Journ.,  (1891)  ii.  1278.  Klein,  ibid., 
(1893)  i.  632,  639,  651.  Klemperer,  Arch.  f.  exper.  Path.  u.  Pharmakol., 
xxxi.  356.  Buchner,  Milnchen.  med.  Wchnschr.,  (1893)  449,  480.  Ehrlich, 
Deutsche  med.  Wchnschr  ,  (1891)  976,  1218.  R.  Pfeiffer,  Ztschr.  f.  Hyg., 
xviii.  i  ;  xx.  198.  Pfeiffer  u.  Kolle,  ibid.,  xxi.  203.  Bordet,  Ann.  de 
rinst.  Pasteur,  ix.  462;  xi.  106.  Metchnikoff,  Virchoirfs  Archiv,  xcvi.  177; 
xcvii.  502;  cvii.  209;  cix.  176;  Ann.  de  rinst.  Pasteur,  iii.  289;  iv.  65;  iv. 
193;  iv.  493;  v.  465;  vi.  289;  vii.  402;  vii.  562;  viii.  257;  viii.  529;  ix.  433. 
Calmette,  Ann.  de  VInst.  Pasteur,  viii.  275  ;  xi.  95.  Fraser,  Proc.  Roy.  Soc. 
Edin.,  xx.  448.  Marmorek,  Ann.  de  PInst.  Pasteiir,  ix.  593.  Metchnikoff, 
Roux,  et  Taurelli-Salimbeni,  ibid.,  x.  257.  Charrin  et  Roger,  Compt.  rend. 
Soc.  de  biol.,  (1887)  667.  Gruber  u.  Durham,  Munchen.  med.  Wchnschr., 
(1896)  March.  Durham,  Journ.  Path,  and Bacteriol.Av .  13.  Cartwright  Wood, 


550  BIBLIOGRAPHY. 

Lancet,  (1896)  i.  980  ;  ii.  1 145.  Sidney  Martin,  "  Serum  Treatment  of  Diphthe- 
ria," Lancet,  (1896)  ii.  1059.  Ransome,  "On  Immunity  to  Disease,"  London, 
1896.  Burdon  Sanderson,  "Croonian  Lectures,"  Brit,  Med.  Journ. ,  (1891)  ii. 
983, 1033,  1083,  1 135.  Discussion  on  Immunity,  Path.  Soc.  London,  Brit.  Med. 
Journ.,  (1892)  i.  373.  Fodor,  Deutsche  med.  Wchnschr.,  (1887)  No.  34. 
Hueppe,  Berl.  klin.  Wchnschr.,  (1892)  No.  17.  Nicholle,  Ann.  de  rinst.  Pas- 
teur, xii.  161.  Salomonsen  et  Madsen,  ibid.,  xi.  315;  xii.  763.  Roux  et 
Borrell,  ibid.,  xii.  225.  Salimbeni,  ibid.,  xi.  277.  Wassermann  u.  Takaki, 
Berl.  klin.  Wchnschr.,  (1898)  xxxv.  4.  Blumenthal,  Deutsche  med.  Wchnschr., 
xxiv.  185.  Ransom,  ibid.,  xxiv.  117.  Meade  Bolton,  Journ.  Exper.  Med.,  i. 
543.  Fraser,  T.  Rt,  Brit.  Med.  Journ.,  (1895)  i.  1309;  ii.  415,  416;  (1896)  i. 
957;  (1896)  ii.  910;  (1897)  ii.  125,  595.  Calmette,  Ann.  de  rinst.  Pas- 
teur, vi.  160,  604;  viii.  275;  ix.  225;  x.  675;  xi.  214;  xii.  343.  C.  J. 
Martin,  Journ.  Physiol.,  xx.  364;  Proc.  Roy.  Soc.  London,  Ixiv.  88.  C.  J. 
Martin  and  Cherry,  ibid..  Ixiii.  420.  Gautier,  "  Les  Toxines  microbiennes  et 
animales,"  Paris,  1896.  Wassermann,  Berl.  klin.  Wchnschr.,  (1898)  1209. 
Pfeiffer  u.  Marx,  Ztschr.f.  Hyg.,  xxvii.  272.  Bordet,  Ann.  de  rinst.  Pasteur, 
xii.  688.  Ehrlich,  Deutsche  med.  Wchnschr.,  (1898)  xxiv.  597.  "Die  Wert- 
bemessung  des  Diphtherieheilserums,"  Jena,  1897;  Croonian  Lecture,  Proc. 
Roy.  Soc.  London,  Ixvi.  424;  Deutsche  med.  Wchnschr.,  xxvii.  (1901)  866,  888, 
913;  NothnagePs  "  Specielle  Pathologic  und  Therapie,"  Bd.  viii.  Schlussbe- 
trachtungen.  Ehrlich  u.  Morgenroth,  Berl.  klin.  Wchnschr.,  (1899)  xxxvi. 
6,  481  ;  (1900)  xxxvii.  453,  681  ;  (1901)  xxxviii.  251,  569,  598.  Weigert,  in 
Lubarsch  u.  Ostertag,  "  Ergebnisse  der  Allgemeinen  Pathologic,"  (1897)  iv. 
Jahrg.,  (Wiesbaden,  1899).  Morgenroth,  Centralbl.  f.  Bakteriol.  u.  Parasi- 
tenk.,  xxvi.  349.  Bulloch,  Trans.  Jenner  Inst.,  2nd  ser.  p.  46.  Donitz, 
Deutsche  med.  Wchnschr.,  (1897)  xxiii.  428.  Bordet,  Ann.  de  rinst.  Pasteur, 
xii.  688;  xiii.  225,  273;  xiv.  257;  xv.  303.  Metchnikoff,  ibid.,  xiii.  737;  xiv. 
369;  xv.  865.  Gengou,  ibid.,  xv.  232.  Sawtschenko,  ibid.,  xvi.  106.  Ritchie, 
Journ.  of  Hyg.,  ii.  215,  251,  452  (with  full  references).  MetchnikofF,  "  L'im- 
munite  dans  les  maladies  infectieuses,"  Paris,  1901.  Neisser  u.  Wechsberg, 
Miinchen.  med.  Wchnschr.,  1901,  No.  18.  Von  Dungern,  ibid.,  (1899)  1288; 
(1900)  677,  963. 

APPENDIX   A.  — SMALLPOX. 

Jenner,  "  An  Inquiry  into  the  Causes  and  Effects  of  the  Variola  Vaccinae," 
London,  1798.  Creighton,  art.  "Vaccination"  in  Encyc.  Brit.,  9th  ed. 
Crookshank,  "Bacteriology  and  Infective  Diseases."  McVail,  "Vaccination 
Vindicated."  Chauveau,  Viennois  et  Mairet,  "Vaccine  et  variole,  nouvelle 
etude  experimentale  sur  la  question  de  1'identitd  de  ces  deux  affections,"  Paris, 
1865.  Klein,  Rep.  Med.  Off.  Loc.  Govt.  Board,  (1892-93)  391  ;  (1893-94) 
493.  Copeman,  Brit.  Med.  Journ.,  (1894)  ii.  631  ;  Journ.  Path,  and 
BacterioL,  ii.  407 ;  art.  in  Clifford  Allbutt's  "  System  of  Medicine,"  vol.  ii. 
L.  Pfeiffer,  "  Die  Protozoen  als  Krankheitserreger,"  Jena,  1891.  Ruffer,  Brit. 
Med.  Journ.,  (1894)  June  30.  Beclere,  Chambon,  et  Menard,  Ann.  de 
rinst.  Pasteur,  x.  i;  xii.  837.  Copeman,  "Vaccination,"  London,  1899. 
Van  der  Leff,  ref.  in  Zeit.f.  Hyg.  u.  Infektskr.,  (1901)  xxxviii.  L.  Pfeiffer, 


BIBLIOGRAPHY.  551 

ibid.,  (1887)  iii.  214.  Guarnieri,  Arch,  per  le  sc.  med.  (1892),  xvi. ;  Clinic. 
Modern.,  (1897)  iii.  Wasielewski,  Zeit.f.Hyg.  u.  Infektskr.,  (1901)  xxxviii. 
212.  Gorini,  Centralbl.  f.  Bakt.  u.  Parasitenk.,  Abt.  i.  (1902),  Bd.  xxxii. 
in,  213. 

APPENDIX   B.  —  HYDROPHOBIA. 

Pasteur,  Comfit,  rend.  Acad.  d.  sc.,  xcii.  1259;  xcv.  1187;  xcviii.  457, 
1229;  ci.  765;  cii.  459,  835;  ciii.  777.  Schaffer,  Ann.  de  VInst.  Pasteur, 
iii.  644.  Fleming,  Trans,  jth  Internat.  Cong.  Hyg.  and  Demog.,  iii.  16. 
Helman,  Ann.  de  VInst.  Pasteur,  ii.  274;  iii.  15.  Babes  et  Lepp,  ibid.,  iii. 
384.  Nocard  et  Roux,  ibid.,  ii.  341.  Roux,  ibid.,  i.  87  ;.ii.  479.  Bruschet- 
tini,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  xx.  214;  xxi.  203.  Memmo, 
ibid.,  xx.  209;  xxi.  657.  Frantzius,  ibid.,  xxiii.  782;  xxiv.  971.  Van 
Gehuchten,/£wr#.  de  Neural.,  (1900)  v.  369,  389.  Van  Gehuchten  et  Nelis, 
Nevraxe,  (1900)  i.  79,  114.  Ravenel  and  McCarthy,  Univ.  Med.  Mag., 
(Philadelphia)  1901,  Jan.  Spiller,  ibid. 

APPENDIX   C.  —  MALARIAL  FEVER. 

Laveran,  Bull.  Acad.  de  med.,  (1880)  ser.  n.  ix.  1346;  "Traitd  des  fievres 
palustres,11  Paris,  1884;  "  Du  paludisme  et  de  son  hematozoaire,"  Paris,  1891. 
Marchiafava  u.  Celli,  Fortschr.  d.  Med.,  1883  and  1885  ;  also  in  Vir chow's 
Festschrift.  Golgi,  Arch,  per  le  sc.  med.,  1886  and  1889  ;  Fortschr.  d.  Med., 

(1889)  No.  3  ;  Ztschr.f.  Hyg.,  x.  136;  Deutsche  med.  Wchnschr.,  (1892)  663, 
685,  707,  729;  Sternberg,  New  York  Med.  Rec.,  xxix.  No.  18.     James,  ibid., 
xxxiii.  No.  10.     Councilman,  Fortschr.  de  Med.,  (1888),  Nos.  12,  13.     Osier, 
Trans.  Path.  Soc.  Philadelphia,  xii.  xiii.     Grassi  and  Feletti,  Riforma  med., 

(1890)  ii.  No.  50.     Canalis,  Fortschr.  d.  Med.,  (1890),  Nos.  8, 9.     Danilewsky, 
Ann.  de  VInst.  Pasteur,  xi.  758.     "Parasites  of  Malarial  Fevers,'1  New..Syd. 
Soc.,  1894  (Monographs  by  Marchiafava  and  Bignami,  and   by  Mannaberg, 
with    Bibliography).     Manson,    Brit.    Med.  Journ.,    (1894)    i.    1252,    1307; 
Lancet,    (1895)    ii.   302;    Brit.  Med.  Journ.   (1898),  ii.  849.     Koch,  Berl. 
klin.  Wchnschr.,  (1899)  69.     Ross,  Indian  Med.  Gaz.,  xxxiii.  14,   133,  401. 
448.     Nuttall,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  xxv.  877,  903  ;  xxvi. 
140;  xxvii.  193,  218,  260,  328  (with  full  literature).     Manson,  Lancet,  (1900) 
i.  1417;   (1900)  ii.  151.     Gray,  Brit.  Med.  Journ.,  (1902)  i.  1121.     Leishman, 
ibid.,  (1901)  i.  635  ;  ii.  757.     Daniel,  'ibid.,  (i901)  i-  193-    Celli>  ibid->  (T901)  *• 
1030.     Nuttall  and  Shipley,  Journ.  of  Hyg.,  i.  45?  269>  45 1  (with  literature). 
Ross,  Nature,  Ixi.  522;    "Mosquito  Brigades  and  how  to  organise  them,11 
London,  1902.     Celli,  "Malaria,11  trans,  by  Eyre,  London,  1900.     Lankester, 
Brit.  Med.  Journ.,  (1902)  i.  652. 

APPENDIX   D.  — DYSENTERY. 

Losch,  Virchow^s  Archiv,  Ixv.  196.  Cunningham,  Quart.  Journ.  Micr. 
Sc.,  N.S.  xxi.  234.  Kartulis,  Virchow^s  Archiv,  cv.  118;  Centralbl.  f.  Bak- 
teriol. u.  Parasitenk.,  ii.  745  ;  ix.  365.  Koch,  Arb.  a.  d.  k.  Gsndhtsamte.,  iii. 
65.  Councilman  and  Lafleur,  Johns  Hopkins  Hosp.  Rep.,  (1891)  ii.  395. 


552  BIBLIOGRAPHY. 

Maggiora,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  xi.  173.  Ogata,  ibid.,  xu 
264.  Schuberg,  ibid.,  xiii.  598,  701.  Quincke  u.  Roos,  Berl.  klin.  Wchnschr., 
(1893)  1089.  Kruse  u.  Pasquale,  Ztschr.  f.  Hyg.,  xvi.  i.  Ciechanowski 
u.  Nowak,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  xxiii.  445.  Howard  and 
Hoover,  Am.Journ.  Med.  Sc.,  (1897)  cxiv.  150,  263. 


INDEX. 


241, 


Abrin,    .... 
"       immunity  against, 
Abscesses,  bacteria  in, 
"         in  dysentery, 
Absolute  alcohol,  fixing  by, 
Acid-fast  bacilli,    . 

"  bibliography, 
"  characters  of, 
"  stain  for, 

Acids,  formation  in  ordinary  media 
Acquired  immunity  fn  man, 

theories  of,   . 

Actinomyces 

characters  of,    . 
"  cultivation  of,  . 

inoculation  with, 
varieties  of, 
Actinomycosis,      .... 

bibliography, 
-"  diagnosis  of, 

-  "  lesions  in, 

origin  of, 

Active  immunity 

Aerobes 

culture  of,         ... 
separation  of,  . 

Aerogenes  capsulatus,  bacillus,  186, 
^Bstivo-autumnal  fevers, 
Agar  media,  ..... 

separation  by,     . 

Agglutination,       .... 
B.  dysenteriae, 
B.  mallei, 
B.  typhosus,  etc., 
bibliography,  . 
cholera  vibrio, 
glanders  bacilli, 
M.  melitensis, 
methods, 
plague  bacillus, 
relapsing  fever, 
serum,      .       109,  344, 
theories  regarding, 
tubercle  bacillus, 
yellow  fever  bacillus, 
Air,  bacteria  in,     .        .        ... 

bibliography, 

Albumose  of  anthrax,  immunity  by 
Albumoses 


PAGE 

.     177 

468,  473 

.     181 

.       92 
254-  256 

•  542 

•  255 
.     104 

,   •  327 

.  461 

.  489 

.  16 

.  287 

•  294 
.  296 

•  295 
.  287 

•  543 
.  296 

291,  292 

•  293 
462,  464 

.   19 
.   49 

•  53 
402,  547 

•  525 
38,40 

•  57 

•  484 

•  352 

•  283 

•  33° 

•  537 

•  423 

•  283 

.  109 

•  446 

•  45i 
454-  480 

•  485 
.  266 

•  457 
.  123 

•  537 
i  •  3J3 

.  174 


Albumoses  in  diphtheria, 
in  tetanus,  . 
in  tubercle, 

Alexines, 

Alkaline  blood-serum, 


PAGE 

.  368 

.  386 

.  262 

482, 498 

45 


Allied  streptothrices,  bibliography,  .  543 
Amboceptors,  .....  490 
Amoebic  dysentery,  ....  529 
Amoebulae  of  malaria,  .  .  .  .  520 

Anaerobes 19 

cultures  of 59 

separation  of,  .         .      60 

toxins  of,     .         .         .        .63 
Anaerobic  plate-cultures,  Novy's  and 

Bui  loch's  apparatus  for,    .         .         60,  61 

Anaesthetic  leprosy,       .         .         .     267,  269 

Aniline  oil,  dehydrating  by,          .         .       96 

"         "    water,  '  101 

"       stains,  list  of,  .        .         -97 

Animals,  autopsies  on,          .         .         .     120 

"         inoculation  of,        .         .       58,  117 

"         tuberculosis  in,  .        .     237 

Anthracis,  bacillus,       .        .        .        .    301 

Anthrax, 300 

"        bacillus  of,       ....    301 

"  "        biology  of,         .        .    304 

"         cultivation  of,    .      302,  317 

"         inoculation  with,  309,  318 

"  "         toxins  of,  .         .         .     312 

bibliography 544 

diagnosis  of,    .         .        .        .     317 
"         disposal  of  animals  dead  of,  .     315 

"         history 300 

"         immunising  of  animals,          .     315 

"         in  animals 306 

"        in  man 309 

"         protective  inoculation,    .         .     315 
"        spread  of,         .        .        .        .314 


Anti-abrin,     . 
Anti-anthrax  serum, 
Antibacterial  sera, 
Anticharbonneux  serum, 
Anti-cholera  vaccination, 
Anti-diphtheritic  serum, 
Antikorper,   . 
Anti-plague  sera,  . 

"          inoculation, 
Antipneumococcic  serum, 
Antirabic  serum,  . 


•  473 
.        -     316 

463,  469,  479 

.     420 

'.         '.     462 

•  445 

•  445 
.     220 

5*4 


553 


554 


INDEX. 


PAGE 

Anti-ricin, 473 

Antiseptics,   ......  140 

"         actions  of,   .        .        .        .  141 

"         bibliography,       .        .        .  537 

"          testing  of,    .        .        .        .  140 

Antisera  (summary),    ...        .  487 

"        therapeutic  action  of,     .        .  488 

Antistreptococcic  serum,      .        .        .  479 

Antitetanic  serum 389 

preparation  of,         .  470 

Antitoxins,  action,  nature  of,        .        .  474 

"          bodies  in  normal  tissues,  .  476 

"          chemical  nature  of,    .        .  477 

"          origin  of,     .         .        .        .  475 

"          sera,  use  of,        .        .        .  488 

"         serum,         .        .     463,  469, 470 

"      cholera,    .        .        .  421 

"      standardisation  of,  .  472 

"         side-chain  theory,       .        .  490 

Antitubercular  serum 264 

Antityphoid  serum 347 

Appendicitis,  .....  194 
Arnold  steam  steriliser,  .  •  .  .30 
Arthrospores,  question  of  occurrence 

of, 8, 409 

Artificial  immunity,  varieties  of,  .        .  462 

Ascomycetae :  oidium  lactis,        .        .  149 

Aspergillus  niger,  form  of  fungi,          .  150 

Attenuation  of  virulence,      .        .        .  464 

Autoclave 31 

Autopsies  on  animals,           .        .         .  120 

Avian  tuberculosis,       .         .        .     238,  252 

Bacilli,  see  Acid-fast. 

Bacillus  acidi  lactici,  .  .  .  .22 
acnes,  .  .  .  .  .188 
"  aerogenes  capsulatus,  .  186, 402 
"  anthracis,  ....  401 
"  botulinus,  .  .  .  398,  547 
"  coli  communis,  lesions  caused 

by 186,  192 

"         "   characters  of,  .        -325 

"         "    comparison  with  B.  typho- 

sus 326 

"  "  detecting  in  water,  .  .  136 
"  "  in  soil,  .  .  .  .131 
"  "  pathogenicity  of,  .  .  338 
"  diphtherise,  ....  357 
"  diplo-bacillus  of  conjunctivitis,  202 
"  dysenteriae,  .  .  .  350,  354 
"  enteriditis  (Gaertner),  .  .  331 
"  enteriditis  sporogenes,  .  .  354 
"  "  "  in  soil,  .  131 

"       of  glanders,      .         .         .     275,  277 
"       icteroides,        ....    456 
"       of  influenza,  see  that  title. 
"       of  Koch-Weeks,      .         .         .     201 
lactis  aerogenes,      .         .        .     186 
of  leprosy,        ....     269 
"       of  malignant  oedema,      .        .    394 


PAGE 

Bacillus  of  Miiller,        ....    201 
"       mycoides  in  soil,     .        .        .     130 

"       Neapolitanus 319 

"       ozcenae 286 

"       ofparacolon,    ....     331 
"       of  plague,  see  that  title. 

"       pneumoniae 207 

"  pseudo-diphthericus,  .  .  370 
"  psittacosis,  .  .  .  .331 
"  pyocyaneus,  ....  186 
agglutination  of,  .  484 
occurrence  of,  .  194 
"  pyogenes  fcetidus,  .  .  .  182 
11  of  quarter-evil,  .  .  .  401 
"  of  rhinoscleroma,  .  .  .  284 
"  of  smegma,  .  .  .  234,  256 
"  of  soft  sore,  .  .  .  .  23 1 

"       species 14 

"       subtilis,     .        .        .  .58 

syphilis,    .....     233 
"       of  tetanus,  see  that  title. 
14       of  Timothy  grass,    .         .         .     255 
"       of  tubercle,  see  that  title. 
"       of  typhoid  fever,  see  that  title. 
"       xerosis,     .....     373 
"       of  Welch,         .         .         .     402,406 
Bacteria,  action  of,         .        .        .       23,161 
"        aerobic  (see  Aerobes) ,  .        .19 
"         air,  see  that  title. 
"        anaerobic  (see  Anaerobes),  .       19 
"        biology  of,       .        .        .        .17 

"        capsulated 4 

"         chemical  action  of,          .       23,  162 

"        composition  of,       .       10 

"        classification  of,      .        .        .  i,  n 

"        cocci,  forms  of,       .        .        .       12 

counting  of  colonies,      .         .      71 
"         cultivation  of,          ...       27 
"         culture  media,  see  that  title. 
"         death  of,          ....     140 

disease,  relation  of  bacteria 

to 157 

"         effects  of  action,     .         .         .     164 

"         ferments,  see  that  title. 

"        food  supply  of,  .         .       17 

gaseous  environment,    .         .       19 

growth,  1         .         .17,  115 

"         higher,  see  that  title. 
"         indol,  formation,    .         .        80,  328 
"         inflammation,  caused  by,       .     182 
"         light,  effect  of,         ...       20 
"         lower,  see  that  title. 
"         metabolism,  disturbances  by 

bacteria,      .        .        .     164,  169 

microscopic  examination 

of,        ....      87,  115 
"         moisture  for,  .  .         .18 

"        morphological  relations  of,    .        2 
"         motility  of,  .         .         .8,  21 

"        movements  of,        .        .        .21 


INDEX. 


555 


Bacteria,  multiplication  of,  .  .  .  4 
"  nature,  effects  in,  .  .  22,  25 
"  nitrifying,  ....  25 

parasitic 23 

pathogenic,  action  of,    .        .     157 
effects  of,     .         .     164 
"         reproduction,          ...        4 
"         saprophytic,    .         .         .23,  157 
"         separation  of,          ...       53 
"        signs  of  infection,  .        .        .171 
"         soil,    examination   for  bac- 
teria  128 

"         species  of,       .         .         .         .11 
"        spore  formation  in  (see  also 

Spores) 5,  58 

"         staining  methods,  see  that  title. 
"        structure  of,    .         .         .         .        3 
sulphur-containing,        .         .       10 
"         suppuration,  see  that  title. 
"        temperature  of  growth  of,      .       19 
"         thermal  death-point  test,        .       70 
"         tissue  changes,        .        .        .     164 
"         toxins,  see  that  title. 
"        variability  among,  .         .       25 

"         virulence  of,    .         .        .     158,  466 
k"         water,  bacteria  in,  .         .     133 

"         see  also  names  of  diseases. 
Bacterial  ferments,        .        .         .        24,  78 

pigments 10 

protoplasm,  structure  of,        .        9 
treatment  of  sewage,      .        .     138 
Bactericidal  powers  of  serum,      .         .    497 
substances,        .         .         .481 
Bacteriological  diagnosis,     .        .         .112 
examination  of  dis- 
charges,    .        .         .114 
Basic  aniline  stains,      ....      97 
Beerwort,  culture  media,      ...      49 
"         gelatin,  ....       41 

Beggiatoa, 16 

Behring  on  immunity,  .  .  .  390,  498 
Bettencourt's  spirillum,  .  .  .  425 
Bibliography,  .....  534 
Biology  of  bacteria,  .  .  17 

bibliography,      .    535 
Bismarck-brown,          ....      97 

Blackleg 4°J 

Blastomycetic  dermatitis,  .  .  .  152 
Blastophores  (malaria),  .  .  .  522 

Blood-agar, 42 

Blood,  examination  of,          .         .         72, 90 

in  malarial  fever,       .        .        .516 

in  relapsing  fever,      .         .         .     447 

"       serum,  as  medium,   .        .         43, 45 

"      toxin  in  the  circulating  blood,      387 

Bone-marrow  in  leucocytosis,      .        .     165 

Bordet's  phenomenon,          .         .        .481 

Botulism,  bacillus  of,   .         .        .         .     398 

Bouillon  (see  also  Culture  media),       .       35 

Bovine  tuberculosis 250 


Bread  paste, 
Brieger  and  Boer, 

Fraenkel, 

Bruce,  on  malta  fever,  . 
Buboes, 
Bubonic  pest, 
Buchner  on  alexines,    . 
"          antitoxin,   . 


PAGE 

.      49 

174,  368 

172,  384 

•  452 
228,  233 

•  435 
.     498 

•  475 


Bulloch's  apparatus  for  anaerobic 

culture 61 

Biitschli  on  bacterial  structure,    .        .  10 

Butter  bacillus, 256 

Calmette,       .        .        .      444, 468, 473, 475 

Canon  on  influenza,     .         .        .     430,  433 

Cantani  on  influenza,  ....  434 

Capsules,  staining  of,    .        .        .        .  106 

Carbol-fuchsin,     .....  102 

"      -gentian-violet,  ....  102 

"      -methylene-blue,        .         .         .  101 

".     thionin-blue,      ....  101 

Carbolic  acid  as  antiseptic,           .        .  146 

Carter  on  relapsing  fever,     .        .        .  449 

Cattle  plague 504 

Celloidin  sacs 66 

Cerebro-spinal  fluid,  examination 

by  lumbar  puncture,  73 
Chamberland  and  Roux,  attenuation 

of  B.  anthracis,           ....  465 
Chamber-land's  filter,    .        .         .        ^74 

Charbon  symptomatique,     .        .        .  401 
Chemical  action  of  bacteria,         .       23,  162 

"         composition  of  bacteria,       .  10 
Chemiotaxis,        .        .          21,  181,  494, 498 

Chlorine  as  antiseptic,           .         .        .  143 

Cholera 4°7 

"         bibliography,          .        .         .  547 

"         immunity  against,          .         .  420 

"         methods  of  diagnosis  of,       .  426 
"        preventive  inoculation 

against,       .        .        .        .421 

"         -red  reaction,         .        .         .  412 

sicca 408 

Cholera  spirillum 407 

characters  of,  .        .  408 

"            "           distribution  of,         .  410 
"            "           inoculation 

with,    .        .      415,  420 
"  "  methods  of 

distinguishing,  422 
powers  of  resist- 
ance of,        .         .  413 
"            "           relations  to 

disease,        .        .  423 

"           spirilla  resembling,  424 

"            "           toxins  of,          .        .  417 

Cladothrices  in  soil 13° 

Cladothrix 16 

astevoides,          .        .        .  295 

Classification  of  bacteria,     .        .        .  i,  n 


556 


INDEX. 


J 

AGE 

, 

PAGE 

Clearing  of  sections  

96 

Cultures,  filtration  of,  

74 

Clubs  in  actinomyces  

290 

from  organs,          .         .113 

,  121 

Cocci,  characters  of,     . 

12 

"         fungi,  see  that  title. 

Collodion  sacs,      ..... 

66 

hanging  block, 

70 

Colonies,  counting  of,           ... 

7i 

hanging-drop,  aerobic, 

68 

Comma   bacillus,    (see   also    Cholera 

anaerobic, 

69 

spirillum)  

407 

"        incubation  of,        ... 

82 

Commission  on  tuberculosis, 

260 

"        microscopic  examination 

"              vaccination, 

503 

of,       

87 

Complement,         ..... 

482 

"         permanent  preservation  of,  . 

85 

2OI 

plate-cultures,  see  that  title. 

bibliography, 

539 

"        pure,      ..... 

49 

Copeman  on  smallpox, 

CQC; 

"        "shake,"        .... 

78 

Cornet's  forceps,            .... 

O    3 
89 

sterilisation,  see  that  title. 

Corrosive  sublimate,  as  antiseptic, 

144 

"         storing,           .... 

82 

"          fixing  by,     . 

92 

tubes  

49 

films  of  blood,  etc., 

90 

"         typhoid  bacillus,   .           320  ei 

seq. 

Councilman   and   Lafleur  on 

Curdling  of  milk  by  B.  coli, 

327 

dysentery,           ... 

531 

Cutting  of  sections,      .... 

93 

Counting  of  colonies, 

71 

Cystitis,        .....      194 

,  228 

Cover-glasses,  cleaning  of,   . 

89 

Cytases  482 

-494 

Cowpox,  relation  to  smallpox,      . 

503 

Cytolytic  sera,      

484 

Crescentic  bodies  in  malaria, 

518 

Cultivation,  of  bacteria, 

27 

Danubicus  vibrio,        .... 

424 

bibliography,     . 

536 

Death  of  bacteria,        .... 

140 

Culture  media,  preparation  of: 

"     -point  test,  thermal,     . 

70 

agar,       .... 

38 

De  Bary,  definition  of  species,     . 

25 

"    alkaline  blood 

De  Christmas,  gonococcus  toxin, 

227 

serum, 

45 

Decolourising  agents, 

99 

"    beerwort,  see  that  title. 

Deep  cultures  

62 

"          "    blood  agar,     . 

42 

Dehydration  of  sections, 

96 

"           "        "      serum,  .         .         43 

,45 

Delepine  

in 

"           "     bouillon, 

35 

Deneke's  spirillum,      .... 

429 

"     bread  paste,  . 

49 

Diagnosis,  bacteriological,  . 

112 

"     Capaldi's  medium, 

329 

Diphtheria,  

356 

"           "     Eisner's  medium,  . 

48 

"           diagnosis  of,      . 

374 

"           "     glucose  agar, 

40 

"            immunity  against,     . 

470 

"           "           "        broth, 

38 

origin  and  spread  of, 

373 

"       gelatin, 

4i 

paralysis  in, 

357 

"          "     glycerin  agar, 

40 

"            results  of  treatment, 

488 

broth, 

38 

Diphtheria  bacillus,  action  of,     . 

373 

"          "     Hiss's  serum  media, 

46 

"       association  with 

"     litmus,   .... 

4i 

other   organ- 

"    Loffler's  serum 

isms, 

360 

medium,     . 

45 

"                  "  .     bibliography, 

545 

"     Marmorek's  serum 

"                  "       characters  of, 

357 

media, 

45 

"       distribution  of, 

358 

"     meat  infusion, 

34 

"       history, 

356 

"      '     "    milk  

48 

"                 "       inoculation  with,  . 

364 

"          "     peptone  broth, 

35 

"                 "       isolation  of, 

375 

"                      "         gelatin,     . 

40 

"                 "       powers  of  resist- 

"         "         solution, 

43 

ance  of,    . 

363 

"    potatoes, 

46 

"        staining, 

363 

"     Proskauer's  medium,    . 

329 

"       toxins  of,      .      173 

,366 

"     reaction, 

36 

"       variations  in 

"     serum  agar,  . 

42 

virulence  of, 

369 

"          "     use  of,    . 

49 

Diplo-bacillus  of  conjunctivitis,  . 

202 

Cultures,  anaerobes,    .... 

59 

Diplococcus,        ..... 

14 

"        collodion  sacs, 

66 

"            endocarditidis  encap- 

"         destruction  of, 

85 

sulatus, 

197 

INDEX. 


557 


PAGE 

Diplococcus,  intracellularis 

meningitidis,    .         .         188 

pneumoniae,        .         .         207 

Disease,  relation  of  bacteria  to,        .         157 

Drying  of  sera,  etc.,  in  vacuo,  .  81 

Ducrey's  bacillus,     .         .  231,  232,  233 


Durham's  fermentation  tubes, 

Dysentery,  amoebic,          .         .         . 

bacillary,         .         .         . 

"          bibliography,  .         .         . 

"          characters  of  amoeba  of, 
Dysentery,  methods  of  examination 


79 
529 
349 
551 
529 

533 


Eberth's  bacillus  .....  319 

Ehrlich  on  ricin  and  abrin,       .         468,  473 

on  toxins,      .         .         178,  385,  478 
"       side-chain    theory    of   anti- 

toxin formation,        .        .  490 

Eisner's  medium,       ....  48 

Embedding  in  paraffin,     ...  93 

Emmerich's  bacillus,         .         .        .  319 
Empyema,         ....         214,  433 

Endocarditis,  see  Ulcerative  endo- 

carditis. 

Enhaemospores  (malaria),        .         .  519 

Enriching  method,  Schottelius',        .  426 

Enteric  fever  ......  319 

Enteritis,  dysenteric,          .         .         .  532 

Ermengem  on  botulism,            .        .  398 

stain  for  flagella,     .         .  109 

Erysipelas,          .....  199 

Escherich's  bacillus,          .        .         .  319 

Esmarch's  roll-tubes,        ...  60 

Exaltation  of  virulence,     .         .         .  466 

Examination  of  water,       .         .        .  133 

Exhaust-pump,           ....  75 

Exhaustion,  theory  of,                .        .  489 

Exotospores  (malaria),     .        .         .  522 

Extracellular  toxins,           .        .        .  173 

False  membrane,      .        .        .         193,  357 
Farcy  .......        276 

Feeding,  immunity  by,     .        .        .        468 

Fermentation  by  pneumo-bacillus,  .        213 

"  by  bacillus  coli,  .        326 

"  methods  of  observing,          78 

"  tubes,  ...          79 

Ferments  formed  by  bacteria,  24,  78,  176,  495 

in  diphtheria,      .        .         .        368 

"         in  tetanus,  .         .  •      .        386 

Fever  .......        169 

Film  preparations,  dry,     .        .        .88,  107 

"  .  "  wet,     ...          91 

staining  of,          .          98 

Filter,  porcelain,  gelatined,       .         .         174 

Filtration  of  cultures,         ...          74 

Finkler  and  Prior's  spirillum,  .         416,  428 

Fish,  tuberculosis  in,         ...        254 

Fixateurs,  .....        495 


Fixation  of  tissues 

Flagella,  nature  of,    . 

"         staining  of,  ... 

"        tetanus  bacillus, 

"        typhoid  bacillus, 
Flagellated  organisms  in  malaria,    . 
Fliigge,       ...... 

Food  supply  of  bacteria,  . 
Forceps  for  cover-glasses, 
Formol  alcohol,  fixing  by, 
Formalin  as  antiseptic, 
Foth's  dry  mallein,   .... 

Fraenkel's  pneumococcus,  see  Pneu- 
mococcus. 

Frankland 

Fraser,  T.  R.. 


PAGE 

92,95 
8 

I07 

378 

322 

522 

15 

17 

89 

92 

145 

283 


135 

468,  473 
93 


Freezing  microtome,  section-cutting, 
Friedlander's    pneumobacillus,    see 

Pneumobacillus. 

Frisch  on  rhinoscleroma,          .        .  285 

Fuchsin,  carbol-,        ....  102 
Fungi, 

"       ascomycetoe,           .        .        .  149 

"       bibliography,          .         .        .  537 

"       blastomycetic  dermatitis,       .  152 

"       cultural  characters,        .         .  154 

"       examination,           ...  155 

"       isolation 156 

morphology,           ...  153 

"       mucorinae 149 

"       pathogenic  properties,  .        .  155 
"       perisporiaceae,  see  that  title. 

"       reproduction,         .        .         .  148 

"       torulae,  etc.,    ....  152 

Gamaleia,          ....         427, 428 
Gametes  (malaria),  .        .         517,  521 

Gangrenous  emphysema,          .         393,  396 

pneumonia,          .        .  433 

Gas  production  by  B.  coli,        .        .  328 

Gaseous  environment  of  bacteria,  19 

Gas-regulator,            ....  83 

Geissler's  exhaust-pump,           .        .  75 

Gelatin  media,            ....  40 

"            "       separation  by,  .        .  53 

"       phenolated 330 

"       shake  cultures,      ...  78 

Gelatined  porcelain  filter,         .         .  174 

Gentian-violet 101,  102 

Germicides,  see  Antiseptics. 

Geryk  pump,     .....  82 

Gibraltar,  rock  fever  of,    .        .         .  451 

Gindha  spirillum 425 

Glanders  bacillus,     .         .        .         275,  277 

"               "        action  on  tissues,  281 

"               "        agglutination,       .  283 

"               "        cultivation,            .  278 

"               "        inoculation,          .  280 
"               "        powers  of  resist- 

"              "        ance,    .        .        .  279 


558 


INDEX. 


PAGE 

Glanders  bacillus,  staining,       .        .  278 

Glanders,  bibliography,     .         .         .  542 

"          diagnosis  of,      .         .         283,  284 

"          in  horses,           .         .        .  276 

"          in  man 276 

"          lesions  in,          ...  280 

spread,  mode  of,       .         .  282 

Glucose  media,          .         .         .       38, 40,  41 

"         broth  and  agar,  .         .    38, 40 

"         potato  as  culture  medium,  47,  243 

Gabbett's  methylene-blue  solution,  .  105 

Golgi  on  malaria 516 

Gonidia,     ......  16 

Gonococcus,  characters  of,       .         .  223 

cultivation,            .         .  224 

distribution,          .         .  227 

"            examination,         .         .  230 

"             inoculation  with,          .  226 

joint  affections,  etc.,    .  229 

plate-cultures,      .         .  226 

relations  to  disease,     .  226 

"             staining,        .         .         .  224 

"            toxins  of,               .         .  227 

Gonorrhoea,       ....         223,  540 

Gonorrhoeal  conjunctivitis,       .         .  229 

"            endocarditis,          .         .  230 

"            peritonitis,     .         .         .  229 

"            septicaemia,  .         .        .  230 

Gram's  method,         ....  102 

"         modifications  of,     .  103 

Grass  bacilli,      .....  255 

Greenfield  on  Anthrax,     .         .         311,  4615 
Growth  of  bacteria,  .        .         .17,115 

Gruber  and  Durham's  phenomenon,  485 

Grubler's  stains 97 

Gulland  (methods),           ...  95 

Haemamceba  danilewski,          .        .  524 

"            malarias,      .        .        .  524 

praecox,        ...  524 

"            relicta,         .        .        .  524 

vivax,           .        .        .  524 

Haematozoon  malariae,     .        .        .  516 

Haemolytic  sera 482 

Haffkine   on   anti-cholera   inocula- 
tion   468 

Haffkine's      inoculation       method 

against  plague,       ....  444 

Hanging-block  cultures,    ...  70 

Hanging-drop  cultures,     ...  68 

"   examination  of,  87 

Hankin, 313 

Hansen,  leprosy  bacilli,    .        .        .  269 

Hardening  of  tissues,         ...  92 

Harris's  collodion  sacs,     ...  67 

Hearson's  incubator,         ...  84 

Heat,  sterilisation  by,         ...  28 

Hesse's  tube 123 

Higher  bacteria i,  15 

"             "          reproduction,         .  5 


PAGE 

Hill's   modification  of  Smith's  fer- 
mentation tube 79 

Hiss's  serum  media,          ...  46- 

Hofmann's  bacillus,           .         .        .  371 

Horsepox,          .....  504 

Houston  on  bacteriology  of  soil,      .  129 

Hueppe 7,  15 

Humoral  theory 490 

Hydrogen,  supply  of,         ...  59 

Hydrophobia,    .....  508 

bibliography,      .         .  551 

diagnosis  of,       .         .  514, 

pathology,           .         .  509 

"  prophylactic  treatment 

of,            ...  512 

virus  of,              .        .  511 

Hypodermic  syringes,       .        .        .  119 


Immune-bodies, 


Immunity, 


origin  of, 
acquired,  theories  of, 


482 
484 
460 

461,  489 

462,  464 
462 

549 
.        468- 

•        463 
.        496 

463,  469 
467 
472 


active, 
artificial, 
bibliography, 
feeding,   . 
"  methods, 

natural,    . 
passive,    . 
toxins,      .         . 
"  unit  of,     . 

"  see  also  names  of  diseases. 

Impression  preparations,  .         .         114 

Incubation  of  cultures,      .         .       82,  83,  84 
Indol,  formation  of,  ...  80,  328 

Infection,  conditions  modifying,       .         158 
nature  of,  ...         158 

Inflammation,  bibliography,     .         .         538 
Inflammatory    conditions     due     to 

bacteria, 182 

Influenza. 430 

"         bacillus,  cultivation  of,     .        431 
"         distribution,         .        432 
"         examination,       .         435 
"  "         inoculation,         .        433. 

"  "         powers  of  resist- 

ance,      .         .        432- 
"          bibliography,    .         .         .         548 
lesions  in,    .         432 
sputum  in,   431-433 

Inoculation,  methods  of,  .        .         118 

of  animals,    .         .         .         117 
"  "         separation  by,       58 

protective,      .         .     468  et  seq. 
syringes  for,  .         118,  119 

Intestinal  changes  in  cholera,  .        .        408 
"  "  amoebic    dys- 

entery,      .        529 
"  bactenal   dys- 

entery,        352,  354 


INDEX. 


559 


Intestinal  changes  in  typhoid  fever, 
"          infection  in  anthrax,    . 
"          infection  in  cholera  (ex- 
perimental), 
Intracellular  toxins, 
Intraperitoneal  injection, 
Intravenous  injection,  . 
Involution  forms  in  bacteria, 
Iodine  solution,  Gram's, 
terchloride, 

as  antiseptic, . 
lodoform  as  antiseptic, 

Issaeff, 

Ivanoff ' s  vibrio,    . 


Jenner  on  vaccination, 
Joints,  gonococci  in,  . 
Joseph  —  syphilis  bacilli, 


PAGE 
332 
.  312 

•  415 

•  173 
.  118 
.  119 

5 

.   IO2 
466,  47I 

•  143 
.  147 
.  469 
.  424 

.   501 
.   229 

•  235 


I    Leprosy,  relation  of  the  bacilli  to, 

I      T  .pntnfhriv 


Kanthack  on  madura  disease,     .         .     299 

Kipp's  apparatus,          ....      59 

Kitasato  on  bacillus  of  influenza,         .     430 

ofplague,     .         .     435 

of  tetanus,  377,  383-389 

Klebs-Loffler  bacillus,          .        .        .356 

Klein 364-365 

Klemperer  on  pneumonia,  .        .     220 

Koch  on  avain  tuberculosis,         .         .     252 

"      bacillus  of  malignant  oedema,    .     393 

"      bovine  tuberculosis,    .         .         .     251 

cholera  spirillum,        .        .        .    407 

"      leveller  for  plates,  55 

steam  steriliser,  ....      30 

tubercle  bacillus,         .         .     236,  237 

tuberculin 260 

"  tuberculin  O,"  and  "  R,".         .     263 
Koch- Weeks  bacillus,  .        .        .     201 

Korn's  acid-fast  bacillus,  .  .  .  256 
Kruse  and  Pasquale  on  dysentery,  .  529 
Kuhne's  methylene-blue,  .  .  .  101 


modification 
method, 


of     Gram's 


104 


Laboratory  rules,           ....  85 

Lamb  on  relapsing  fever,     .         .        .  450 

Laveran's  malarial  parasite,         .         .  516 
Leishman,  staining  of  malarial  parasite, 

527-528 

Lenses,           ......  87 

Lepra  cells, 268 

Leprosy 267 

bacillus,          ....  269 

distribution  of          .  271 


staining,  . 
bibliography, 
diagnosis  of,  . 
etiology  of,     . 
forms,     . 
inoculation,    . 
pathological  changes, 


105,  270 
•  542 

270,  274 
.  267 
.  267 
.  272 
.  267 


Leptothrix, 

Lesions  produced  by  bacteria, 

Leucocylosis, 

Leucomaines, 

Light,  effect  on  bacteria, 

Litmus  media, 
whey, 

Liver  abscess  in  dysentery,  . 

Lockjaw 

Loffler's  bacillus, . 
"        flagella  stain, 
"        glanders  bacillus,  . 
"        methylene-blue, 
"        serum  medium. 

Losch ,  amoeba  of, 

Lower  bacteria,     . 

"         reproduction, 

Lumbar  puncture, 

Lustgarten's  bacillus,    . 

Lustig's  anti-plague  serum, 

Lymph,  vaccine,   „ 

Lymphangitis, 

Lysogenic  action  of  serum, 

toward  blood 
corpuscles, 
"  "       theory  of,  . 


PAGE 
.  272 
.  16 
.  164 
165,  495 
.  172 

20 

•  41 
.    41 

•  531 

•  376 

•  356 

.  108 

•  275 

.   100 

•  45 

•  529 

.  I,  12 

4 

•  73 

•  234 

•  445 
.  502 

•  193 
.  480 

.  482 
482 


MacConkey's  bile-salt  media,      .         .  137 

M'Fadyean  on  glanders,       .        .         .  283 

Macrocytase,         ....     484,  494 

Macrophages,        .....  493 

Madura  disease,   .....  297 

"        bibliography,     .         .  543 

Malaria,  cycle  in  man,          .        .        .  517 

"      bibliography,         .         .  551 

"      in  mosquito,          .        .  521 

"        prevention  of,          ...  526 

Malarial  fever,  examination  of  blood 

in, 

"  "      malignant,     . 

"  "      mosquitoes  in, 

"        parasite, 


.    526 

•  525 
517-  521 

.    516 

•  517 


inoculation  of, 
"  "        staining  of,  Leish- 

man's  method,    527-528 
"  "        staining  of,  Muir's 

method,         .        .  527 
"              "        staining  of,  Roma- 

nowsky  method,    .  527 

"              "        varieties  of,        .        .  523 

Malignant  oedema,  bacillus  of,     .        .  393 

"              "         bibliography,         .  547 

"              "         diagnosis  of,  .        .  397 

"  "         experimental 

inoculation,        .  396 

"              "         immunity  against,  397 
"          pustule,        .        .        .        .310 

Mallein 283 

Malta  fever, 451 


560 


INDEX. 


PAGE 

Malta  fever,  bibliography,    .        .        .    549 

"          "       methods  of  diagnosis,     .    454 

Mann's  method  of  fixing  sections,      .      95 

Manson  on  malaria,     .        .      516,  517,  527 

Maragliano's  anti-tubercular  serum,    .     264 

Marchiafava  and  Celli  on  malaria,      .    516 

Marmorek  on  streptococci,  .        .     191 

anti-streptococcic  serum,  .    479 

"          serum  media,      ...      45 

Martin,  C.  J.  (toxins),  .         .         .     174 

"       (antitoxins),    .         .         .    474 

Martin,  Sidney,  on  albtimoses,  etc.,     .     174 

(anthrax),      .         .    312 

(diphtheria),       356,  368 

(tetanus),       .        .    385 

Massowah  vibrio,         .        .        .     416, 425 

Measuring  bacteria 116 

Meat  infusion 34 

Meat-poisoning  by  bacillus  botulinus,     398 
"            "          by    Gaertner's    bacil- 
lus  331 

Mediterranean  fever,     .        .        .        .451 
Melitensis,  micrococcus,       .         .        .     452 
Meningitis,  bibliography,      .         .         .     539 
epidemic  cerebrospinal,  188, 200 
"          in  influenza,       .        .        .    433 
"          pneumococci  in,        .        .    214 
Mercury  perchloride  as  antiseptic,      .     144 
Mesenteric  glands  in  typhoid  fever,     .     332 
Metabolism,  disturbances  of,  by  bac- 
teria       164, 169 

Metachromatic  granules,      ...         9 
Metchnikoff  on  cholera  in  rabbits,      .    416 
"    Pfeiffer's  phenom- 
enon,       .         .        .    481 
"  "    phagocytosis  theory,  .    493 

"    relapsing  fever,    .         .     449 
"    spirillum,     .         .         .    427 
Methylene-blue,    ...        97,  100,  101 

Methyl-violet 97 

Micrococci  of  suppuration,          .        .     182 

Micrococcus, 14 

melitensis,       .        .        .    452 

of  gonorrhoea,         .        .     223 

pyogenes  tenuis,     .        .     182 

"  tetragenus,      .        .        .187 

"  "  lesions 

caused  by,     194 

ureas,      ....      24 

Microcytase,          .....    484 

Microphages, 493 

Microscope,  use  of,  .        .        .87 

Microscopic  methods,  bibliography,    .    537 
Microtomes,  .....      93 

Migula, 14,  15 

Mikulicz,  cells  of,  ....  285 
Milk  as  culture  medium,  ...  48 
"  curdling  by  B.  coli,  .  .  .  327 
Moeller's  grass  bacilli,  .  .  .  255 
Moeller's  stain  for  spores,  .  .  .  106 


PAGE 

Moisture  necessary  to  growth  of  bac- 
teria, .......      18 

Mordants,      ......      99 

Morphology i 

bibliography,  .        .        .    535 

Mosquitoes  in  malaria,        .      516, 517,  521 

r61e  in  yellow  fever,  .        .    458 

Motility 8,  21 

Mucorinae,    ......     149 

Muencke's  filter,   .....      77 

Muir's  staining  method,       .        .     106, 527 

Miiller's  bacillus 201 

Mycetoma, 297 

Mycoderma,  fungi 152 

Myelocytes,  neutrophile,       .        .        .     165 

Nascent  oxygen  as  antiseptic,      .        .     144 
Natural  immunity,         ....    496 

Nature,  effects  of  bacteria  in,      .         22,  25 
Neapolitan  fever, .         .         .        .         .451 

Neelsen's  stain  for  tubercle,         .         .     104 
Neisser's  gonococcus,  .        .        .    223 

Nencki,          ......       n 

Neutral-red,  use  of,  .        .      42,  137 

"          "       "     with  B.  typhosus,      .    329 

Neutrophile  leucocytes,        .        .        .     165 

myelocytes,       .         .         .     165 

Nicolaier,  tetanus  bacillus,  .        .        .    376 

Nicolle's     modification     of     Gram's 

method,      ......     103 

Nikati  and  Rietsch  (cholera),      .        .    415 
Nitrifying  bacteria,        ....      25 

Nitroso-indol  body 81 

Nordhafen  vibrio 428 

Obermeier's  spirillum,           .         .        .  447 
(Edema,   malignant,  see    Malignant 

oedema. 

Ogata's  dysentery  bacillus,  .        .        .  354 

Ogston,          ......  182 

Oidium  lactis,       .....  149 

Oil,  aniline,  for  dehydrating,  etc.,        .  96 

"     immersion  lens 87 

Organisms  lower  than  bacteria,  .         2,  458 

Oriental  plague,    .....  435 

Osteomyelitis 199 

Otitis 214 

Oxygen,  nascent,  as  antiseptic,    .        .  144 

Ozoena  bacillus, 286 

Paracolon  bacillus,       .        .        .        .331 

Paraffin  embedding 93 

"       sections, 

"  "        cutting,      ...      94 

preparation  for 

staining,         .        .      95 
Park's  method  of  anaerobic  culture,   .      64 
Passage,         ......     466 

Passive  immunity,         .        .         .     463, 469 
Pasteur    on    exaltation   of    virulence 
of  bacteria,        .'....    466 


INDEX. 


56l 


PAGE 

Pasteur  on  hydrophobia,      .        .        .  512 
"         on   vaccination   against    an- 
thrax   315 

septicemie  de,  393 
Pathogenic  bacteria,     .        .        .        .158 

Penicillium  glaucum 151 

Peptone  broth  or  bouillon,   ...  35 

"        gelatin 40 

"         solution 43 

Perchloride  of  mercury  as  antiseptic,  144 

Periostitis,  acute  suppurative,       .        .  199 
Perisporiaceae, 

aspergillus  niger,  .        .  150 

penicillium  glaucum,    .  151 

Peritonitis 191,  229 

Perlsucht, 238 

Pestana  spirillum 425 

Pestis  major, 441 

"      minor,         .....  441 

Petri's  acid-fast  bacillus,       .        .        .  256 

"      dishes,        .....  54 

"      sand-filter  for  examining  air,     .  125 

Petruschky's  litmus-whey,    ...  41 

Pettenkofer  on  cholera,         .         .     420,  424 

Pfeffer, 21 

Pfeiffer  on  anti-serum,          .         .        .481 
"          cholera,       .        .         .     417,  422 

"          influenza 430 

"         protozoa  in  smallpox,         .  506 

"          typhoid,       ....  340 

Pfeiffer's  phenomenon,         .        .     422, 481 

Phagocytes, 165 

Phagocytosis  theory,     .         .      490,  493,  494 

Phenol-phthaleine  as  indicator,   .         .  36 

Phenomenon  of  Bordet,        .         .         .  481 

"            "  Gruber  and  Durham,  485 

"  "  Pfeiffer,      .        .     417, 481 

Pigments,  bacterial,  n 

Piorkowski,  syphilis  bacilli,          .         .  235 

Pipettes 113 

Pitfield's  flagella  stain,  .        .        .108 

Plague,  bacillus  of,       ....  435 
anatomical  changes 

and  distribution,  439 

"            "            characters,       .        .  436 

"            "            cultivation,      .        .  438 

"             "            involution  forms,    .  437 
"        ,    "            stalactite      growths 

of,        .        .        .  438 

bibliography,  .         .  548 

"       Haffkine's  inoculation  against,  444 

"       immunity  against,    .         .        .  444 

infection  in,      .        .         .         .  442 

preventive       inoculation 

against,          ....  444 

serum  diagnosis,     .        .        .  446 

"       toxins,       .....  /]/|4 

varieties  of,               .        .        .  439 

Plasmodium  malariae,          .        ,        .  516 

Plasmolysis, 9 

2O 


PAGE 

Plate-cultures,  agar 57 

anthrax,         .        .         .     302 

"          gelatin 53 

"          gonococcus,  .        .        .    226 

Platinum  needles,          ....      51 

Pneumobacillus  (Friedlander's),      207,209 

cultivation,         .         .     212 

effects,      .          .         .     218 

examination,      .     209,  222 

Pneumococcus  (Fraenkel's),       .     207,  208 

cultivation,  .         .     210 

examination,        .     207,  222 

immunity  against,       .     219 

in  endocarditis,  .     197 

inoculation,          .        .     215 

lesions  caused  by,       .     213 

toxins  of,     .         .        .     219 

Pneumonia,  bacteria  in,       ...     207 

bibliography,    .         .         .     539 

gangrenous,      .         .     213,  433 

history,      ....     206 

in  influenza,      .         .         .     432 

methods  of  examination,       221 

septic 206 

staining  films,  .         .        .     208 
varieties  of,  .         .     205 

Pneumoniae,  bacillus,  ....  207 
diplococcus,  .  .  .  207 
streptococcus,  .  .  207 

Polar  granules 9 

Potassium  permanganate  as  antiseptic,     146 
Potatoes  as  culture  material,        .       46,  279 
Poynton   and   Payne   on  acute  rheu- 
matism  203 

Preparations,  impression,     .        .        .114 
Prophylactic   treatment  of  hydropho- 
bia  512 

Protective  inoculation,          .        .        .    467 

Proteosoma 516,  524 

Protozoa  described  in  hydrophobia,.     511 
smallpox,         .    506 

Protozoon  malariae,      ....     516 

Pseudo-diphtheria  bacillus, .        .         .     370 

"       -tuberculosis  streptothricea,      .     295 

Psittacosis  bacillus,       ....     331 

Ptomaines 172 

Puerperal  septicaemia, ....     193 
Pus,  examination  of,     .         .         .       90,  204 

Pustule,  malignant 310 

Pyaemia 193 

"        nature  of,  ...     181 

Quartan  fever 524 

Quarter-evil,  bacillus  of,        ...     401 

bibliography,   .        .         -547 

Quotidian  fever 523 

Rabies,  see  Hydrophobia. 

Rabinowitsch's  acid-fast  bacillus,         .     256 

Rauschbrand  bacillus,          .         .        .    401 


562 


INDEX. 


PAGE 

Ray-fungus  (actinomyces),          .         .  287 

Reaction  of  media,  standardising  of,  36 

Receptors 490 

Recovery  from  disease,       .         .         .  462 
Red  stains,           .  97 
Reichert's  gas  regulator,     ...  83 
Relapsing  fever,  agglutination  of  spiril- 
lum,    .         .         .  451 
"             "    bactericidal  serum  in,  451 
"     bibliography,     .         .  489 
"     spirillum  of,  etc.,       .  447 
Retention,  theory  of,  .         .         .         .  489 
Rheumatism,  acute,   ....  202 
"            bibliography,        .         .  539 
Rhinoscleroma,  bacillus  of,         .         .  285 
bibliography,     .         .  543 

Ricin, 177 

"       immunity  against,    .         .       468,  473 

Rietsch  and  Nikat,  cholera,        .         .  415 

Rivers,  bacteria  in,              .         .         .  135 

Robin,         ......  177 

Rock  fever,         ' 451 

Roll-tubes,  Esmarch's,        ...  56 

Romanowsky,  staining  method,          .  527 

Romanus  spirillum,    ....  425 

Rosenbach  (bacteria  in  suppuration),  182 

Ross  on  malaria,         ....  522 

Roux  on  antitoxic  sera,      .         .         .  472 
"       "    diphtheria,  .          172, 356,  364-371 

Saccharomyces,          ....  152 

Safranin 97 

Salt-agar  as  medium  for  B.  pestis,     .  437 

Sanarelli,  typhoid  fever,      .         .         .  335 

"         yellow  fever,        .         .         .  457 

Sanderson,  Burdon,   .         .         .       465,  505 

Saprophytes 23,  157 

Sarcina,      .         .         .         .         .         .  13,  14 

Sausage  poisoning  by  bacillus  botu- 

linus,       ......  398 

Scarification 118 

Schizomycetes,  ......  3 

Schizophyceae,    .....  3 

Schizophyta,       .....  3 

Schottelius"  enriching  method,  .         .  426 

Schuylkiliensis  spirillum,   .         .         .  428 

Scorpion  poison,         ....  178 

Section-cutting,           ....  93 

"        fixation  methods,  .         .            92, 95 
Sections,  dehydration  of,   .         .         .  96 
Sedgwick-Tucker  method  for  exam- 
ining air, 125 

Sedimentation  methods,    .         .         .  109 

test  for  typhoid,          .  341 

Seitenketten,       .         .         .         .         .  490 

Separation  of  aerobes,  53 

"    anaerobes,  ...  60 

Septicaemia,  nature  of,  .         .181 

puerperal,      .         .         .  193 

"           sputum,          .         .        .  207 


PAGE 

Septicemie  de  Pasteur,       .         .         .  393 

Septic  pneumonia,      ....  206 

Sera,  haemolytic,         ....  482 
Serum,  agar,       .                  .         .         .42 
"       agglutinative  action  of, 

109,  344,  454,  484 

antibacterial,          .         463,  469,  479 

anticharbonneux,  .         .         .  316 

"       anticholera,             .         .         .  420 

"       antidiphtheritic,     .         .         .  470 

"       anti-plague,  see  that  title. 

antipneumococcic,         .         .  219 

antirabic,        ....  514 

antistreptococcic,  .        .  •       .  479 

"       antitetanic 389 

"       antitoxins,  see  that  title. 

"       antitubercular,        .         .         .  264 

"       antityphoid 347 

"       bactericidal  action  of,    .         .  498 
blood,    .....  43,  45 

diagnosis 485 

methods,         .         .  109 
"         of  typhoid,      .         .  340 
"       inspissator,    ....  44 
"       lysogenic  action  of,        .         .  480 
"          towards  blood  cor- 
puscles,      .         .  482 
"       media,   .....  45 
"       sedimenting  properties,          .  109 
Sewage,  bacterial  treatment  of,           .  138 
contamination  of  water  by,  .  137 
Shake  cultures,  .....  78 

Sheep-pox, 505 

Shiga's  bacillus,          ....  350 

Side-chain  theory,  Ehrlich's,       .         .  490 

Singer,  acute  rheumatism,          .         .  203 

Slides  for  hanging-drops,  ...  68 

Sloped  cultures,  aerobic,    ...  50 

anaerobic,        .         .  66 

Smallpox,  ......  501 

bacilli  in 505 

"          bibliography,      .         .         .  550 


micro-organisms     associ- 
ated with. 


505 


protozoa   as   causative 

agents,    ....  506 

"          relation  to  cowpox,    .         .  503 
Smegma  bacillus,       .         .         .       234,  256 

Smith,  T. —  detecting  B.  coli  in  water,  136 
Smith's     fermentation     tube,     Hill's 

modification,  .....  79 

Smith's,  Lorrain,  serum  medium,      .  45 

Snake  poisons,  .         .          178,  468,  473-475 

"        immunity  against,      .  468 

Soft  sore,    .         .        .         .        .         .  231 

1     bibliography,       .         .         .  540 
Soil,  examination  of,  for  bacteria,      .  128 
Soudakewitch  on  relapsing  fever,       .  450 
Spinal  cord,  lesions  by  pyogenic  or- 
ganisms,         .         .         .         .         .  192 


INDEX. 


563 


PAGE 

Spirilla,  characters  of, .        .        .         -15 

like  cholera  spirillum,     .         .  424 

Spirillum  Metchnikovi,        .         .         .  427 

"         of  Deneke 429 

"         of  Finkler  and  Prior,    .     416,  428 

"         of  Miller 429 

"         relapsing  fever,  inoculation 

with,  etc 447 

"         Schottelius'       enriching 

method,     ....  426 

"         Schuylkiliensis,    .         .         .  428 
see  also  Cholera  spirillum  and  Vibrio. 

Spirochasta, 15 

Spleen,  typhoid  bacilli  in,     .         .     333,  348 

Splenic  fever 300 

Spore  formation,  arthrosporous, .         .  7 

"              "           B.  anthracis,     .         .  304 

B.  tetani,  .         .     378,  381 

"              "           endogenous,     .         .  5 

malaria,    .        .         .  519 

Spores,  staining  of,      .         .         .         .  106 

Sporocyst  (malaria),    ....  522 

Sporocytes,  in  malaria,         .         .         .  521 

Sporulation  of  malarial  parasite,          .  521 

Sputum,  amoebae  in,    ....  532 

influenza,        .         .         .     431,  433 

"        in  plague 447 

"  in  pneumonia,  .  .  .  208 
"  phthisical,  .  .  243,  248,  259 

"        septicaemia,    ....  207 

Staining  methods 96 

"        capsules,  Welch's  method,   .  106 

diphtheria  bacillus,         .         .  363 

films 98 

flagella, 107 

formulas   of   stain   combina- 
tions,        ,  100 
"        Gabbett's        methylene-blue 

solution 105 

"        glanders  bacillus,  .         .         .  278 

41  gonococcus,  ....  224 
"  leprosy  bacilli,  .  .  105,  270 
41  malarial  parasite,  see  that 

title. 

Muir's  method,      .         .     106, 527 
"        paraffin     sections,     prepara- 
tion for  staining,         .         .  95 
•*        pneumonia  films,  .         .         .  208 
"        principles,      ....  96 

"        spores 106 

"        tubercle  bacilli,      .         .         .  104 

Ziehl-Neelsen,        .         .         .  104 

Stains,  basic  aniline,    ....  97 

Stalactite  growths,  plague  bacillus,      .  438 

Standard  of  immunity,         .         .         .  472 

Standardising  reaction  of  media,        .  36 

serum,   ....  472 

Staphylococci,  lesions  caused  by,         .  193 

Staphylococcus,   .         .         .  I3 

"  cereus  albus,     .     182,  184 


PAGE 

Staphylococcus,  cereus  flavus,     .      182,  184 

epidermidis  albus,      .     184 

pyogenes  albus,       182,  184 

aureus,        .     207 

characters 

of,  .         .     182 
inoculation 

with,         .    189 
citreus,        .     184 

Steam  steriliser 30,  31 

Stegomyia  fasciata,       ....     459 
Sterilisation  by  heat,    ....       28 
at  low  temperatures,         .       32 
by  steam   at   high   press- 
ure,     ....       31 
Sternberg's   bulb   for   thermal   death- 
point  test, 70 

Stewart's  forceps,          ....       89 
Stone,  —  detecting  B.  coli  in  water,      .     136 

Storing  of  cultures 82 

Streptococci  in  diphtheria,  .         .         .     360 

in  false  membrarie,~  ~"      .     193 

"  lesions  caused  by,   .         .     193 

"  varieties  of,      .         .         .191 

Streptococcus, 12 

brevis 191 

"  conglomeratus,     .      186,  192 

"  erysipelatis,  .        .         .191 

Streptococcus  longus,  ....     191 
"  pneumonias,  .        .     207 

"  pyogenes,  characters  of,     184 

"  "          inoculation 

with,  .        .     190 

"  "          in  soil,  .        .     131 

Streptothrices,  allied  to  actinomyces,  .     295 

bibliography,        .        .    543 

Streptothrix 16 

"  actinomyces,      .         .        .     288 

"          maduras,     ....     298 

Structure  of  bacteria 3 

Subcultures, 51 

Subcutaneous  injection,       .        .        .     118 

Sugar,  fermentation,      .        .        .       78,  326 

Sulphurous  acid  as  antiseptic,      .        .     146 

Suppuration,  bacteria  of,      .  .     182 

"  bibliography,  .        .        .     538 

"  gonococci  in,  .        .        .     228 

"  methods  of  examination 

of 204 

"  nature  of,         .        .         .     180 

"  origin  of,          ...     195 

"  pneumococci  in,     .        .     214 

"  typhoid  bacillus  in,         .     334 

Symptoms  caused  by  bacteria,    .        .     17  ^ 

Syphilis,  bacillus  of,      .        .        .        •     233 

"        bibliography,          .        .        .     541 

Syringes  for  inoculation,       .        .      118,  119 


Tabes  mesenterica, 
Temperature,  growth  of  bacteria, 


259 
19 


564 


INDEX. 


Tertian  fever, 
Test-tubes  for  cultures, 
Tetanolysin,  . 
Tetanospasmin,     . 
Tetanus, 

"        anti-serum  of,  . 


PAGE 
.  524 
.  49 

•  •     385 

•  -     385 

•  •     376 
389  et  seq. 


intracerebral  injec- 
tion of,        .         .     391 
"        bibliography,   ....     546 
immunity  agairfst,    .         .         .     389 
methods  of  examination,         .     392 
treatment  of,    .         .         .     391,  489 
Tetanus  bacillus,   .....     377 
characters,  .        .        .    377 
inoculation  with,         .     383 
"  "       isolation  of,         .        .    379 

"       pathogenic  effects,      .     381 
"       spores  of,     .         .     379,  381 
"  "      toxins  of,     .         .     173, 384 

Tetrads 13 

Theory  of  exhaustion 489 

"       of  phagocytosis,        .         .     490,493 
"       of  retention,       .         .        0         .     489 

"       humeral, 490 

Thermal  death-point  test,     ...      70 
Thionin-blue,        ....       97,  101 

Thiothrix,       ......       16 

Timothy  grass  bacillus,        .         .         .     255 

Tissues,  action  of  bacteria  on,     .         .     164 

examination  of  bacteria  in,    .       91 

"         fixation  of,  .         .         .92 

Tizzoni  and  Cattani  on  tetanus,  .         .    389 

Torulae 152 

Toxalbumins, 172 

Toxic  action,  theory  of,  ...  178 
Toxicity,  estimation  of,  ...  470 
Toxins,  antitoxins,  see  that  title. 

circulating  blood,  toxin  in,      .     387 
"       concentrated,   method  of  ob- 
"  taining,         ....     176 

"       constitution  of,        ...     478 
"       early  work  on,          .         .         .     171 
effects  of,          ....     168 
"       Ehrlich's  theory,      .       178, 478, 490 
immunisation  by,    .         .         .     467 
"       intra-  and  extra-cellular,          .     173 
"       nature  of,         ....     174 
"       non-proteid,     ....     175 
"       production  of,          ...     162 
susceptibility  to,       .         .         .     499 
"        vegetable,         ....     177 
see  also  names  of  diseases,  An- 
thrax, etc. 

Toxoids, 179, 478 

Toxones 179,  478 

Tropical  malaria,         ....     525 

Tubercle  bacillus,         ....     236 

"       action,       .         .      244,  258 

"       avian,        .         .      238,  252 

"       characters,        .         .     238 


PAGE 

Tubercle  bacillus,  cultivation  of,          .     241 

"  "       dead,  action  of,         .     258 

"  "       distribution  of,  .     246 

"       immunity  against,     .     264 

"       in  sputum,  etc., 

243,  248,  259 

"      inoculation  with,       .     249 
"  "       Koch's  discovery,  236,  237 

powers  of  resistance 

of,          ...     243 

"       stains  for,  .         .     104 

"  "       toxins  of,  .         .         .     262 

"        giant-cells,    .         .         .      244,  245 

methods  of  examination  of,  .     264 

Tubercles,  structure  of,         ...     244 

Tubercular  leprosy,      .         .         .      267,  268 

Tuberculin,  diagnosis  of  tuberculosis 

by,        ....     262 

"          Koch's,      ....     260 

"  O  "  and  "  R,"          ..         .     263- 

Tuberculosis, 236 

"  avian,      .        .         .      238,  252 

bibliography,  .  .  .  541 
bovine,  ....  250 
diagnosis  by  tuberculin,  262 

"  history 236 

"  in  animals,      .         .         .     237 

"  in  fish 254 

modes  of  infection,  .  259,  260 
precautions  in  diagnosis 

of,  .        .         .     257 

symptoms,       .         .        .     246 

varieties,          .         .         .     250" 

Tubes,  cultures  in,       .         .         .         .51 

Typhoid  bacillus,         .         .         .     319,  320^ 

"  "        comparison  with  B. 

coli,       .         .         .     326 

"         cultures,    .       321,  .322,  326 

"        examination  for,       .     347 

"  "         history,      .         .         .     319 

"  "         immunity  against,     .     337 

"  "        inoculation  with,       .     334 

"         relationship  to  fever,     339 

"         serum  diagnosis,       .     340- 

"  "         suppurations  in,        .     334 

"  "         toxins  of,  .         .         .     336 

"  "        vaccination  against,      346 

"        fever, 319 

"      bibliography,       .         .     544 
"      pathological    changes 

in,   .        .        .         .     332- 

Ulcerative  endocarditis,       .         .         .  197 

"                "             experimental,  198 

gonococci  in,  230 

Unit  of  immunity,        .         .        ...  472 

Urine,  examination  of,          ...  74 

staining  of  bacteria  in,      .         .  90 

tubercle  bacilli  in,     ,         .     248,  265 

typhoid  bacilli  in,      .         .         .  348- 


INDEX. 


565 


Uschinsky,  me'dium 368 

nature  of  toxins,        .        .  174 

Vaccination,  Jennerian,       .         .         .  501 

nature  of,         ...  506 
see  also  diseases,  Typhoid, 

etc. 

Vacuo,  drying  substances  in,        .         .  81 

Vaillard  on  tetanus,      ....  388 

Van  der  Loeff,  protozoa  in  smallpox,  506 
Van  Ermengem,  see  Ermengem. 

Van  Niessen,  syphilitic  lesions,   .         .  235 

Variola 503 

Vegetable  poisons,  toxins,   .       177,  468,  473 

Venins, 178 

Vibrio, 15 

"      berolinensis,      ....  424 

of  cholera,          ....  407 

Danubicus 424 

Deneke's,           ....  429 

Finkler  and  Prior's,  .        .     416,  428 

Gindha 425 

Ivanoff,     .....  424 

"      Massowah,        .        .        .     416,  425 

Metchnikovi 427 

Nordhafen,        ....  428 

Pestana  and  Bettencourt,           .  425 

Romanus,           ....  425 
"     see  also  Spirillum. 

Vibrion  septique,          ....  393 

Virulence,  attenuation  of,     .         .         .  464 

exaltation  of,                .         .  466 

"          of  bacteria,          .        .        .  '  158 


PAGE 

Water,  bibliography,   ....    537 

contamination  of,  by  sewage,  .     137 

"       examination  of,        ...     133 

"       typhoid  bacilli  in,     .         .         .     349 

Weichselbaum  on  pneumonia,   .         .     207 

Weigert's  method  of  dehydration,       .       96 

modification      of      Gram's 

method 103 

Welch's  gas-bacillus,   ....    402 
staining  method,    .         .         .     106 
Wertheim's  medium,  .         .      224,  225,  226 
Widal  on  serum  diagnosis,  .         .     485 

Winogradski,       .....       25 

Winter-spring  fevers 523 

Woodhead  on  tuberculosis,         .      259,  260 
Woody  tongue,    .  293 

Woolsorter's  disease,  ....     311 
Wright's  method  of  anaerobic  culture,       64 


Xerosis  bacillus,  . 
Xylol,  . 


Yeast,  form  of  fungi 

Yellow  fever 

"        "      bacteria  in, 
"         "      bibliography,    . 
etiology  of,       . 
"      mosquitoes  in  relation  to, 
Yersin,  anti-plague  serum,  . 


373 
96 

152 
455 
456 
5.49 
456 
458 
445 


diphtheria,       .         172,356,364-371 
plague, 435 


Wasielewski,  smallpox, 
Water,  bacteria  in, 


506 
133 


Ziehl-Neelsen  stain, 
Zoogloea, 
Zygote  (malaria), 
Zygotoblasts  (malaria), 


104 

4 

522 
522 


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